A look beneath the surface Cranial nerve development of Nothobranchius furzeri & the EvoDevo of the trigeminal nerve Benjamin Naumann1*, Nils Hartmann2, Peter Warth1, Christoph Englert2, Lennart Olsson1, Peter Konstantinidis3 Friedrich Schiller University, Dept. of Zoology and Evolutionary Biology, 07743 Jena, Germany. 2 Leibniz Institute for Age Research, 07745 Jena, Germany. 3 Virginia Institute of Marine Sciences, College of William and Mary, Gloucester Point, 23062 Virginia, USA. * corresponding author:
[email protected]. 1
The anatomy of the head
AA
NC
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dop
lap
aop cu OC lop lpe
EB
A
A 8dpf
Vop
r4
r5
Yolk Sac
B 9dpf
ima
r7
VII
r8
C 10dpf
X
VIIaX
A 8dpf
A`
Vop
VIIop
MB
VIIaX
MB
IX
X
S1
HB
HB r2
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Eye
Yolk Sac
Vop
VIIop
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Eye
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VIII
Vmm
Xlt
VII
VII
Yolk Sac
S1
Xb2
Vop VIIop
VIIhm
Xb2
VIIhy
B 9dpf
Vop
VIIop
B`
IX VIIaX
Vop
VIIaX
VIIop
X
X Eye
S1 Yolk Sac
C 10dpf
VII
S1 S2
Eye VII
Xb
Xb2 Xb3
Eye
Yolk Sac
S1 S2
Xb3 Xb2 Xb4
VIIopm VIIaX X VIIhm IX
VIIaX
VIIop
X
Slp S1
VIIaX
VIIopm VIIaX X VIIhm IX
Xlt
IX
S3
Shg
bs
VIIop Eye ph
Vop
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li II
Vima
li I V
VIIop(cut)
od IV
Xb2
VII
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b2
Vdople
I
IX
Xlpo
S1
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Slp S2
PFN (cut) Xin
Xlt
aab I VIIhs
Shg
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Yolk Sac
Xb5
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Vman
Vima
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Xb2 VIIhy
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Xb4 Shg
Eye
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Vam
Yolk Sac
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Vima
VIIop(cut)
Vop
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Vdop
Eye
VIIih VIIhi
Vimp
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Xb3
Xlpo
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S1
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Vimp
VIIhm Vman
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VIIhs
Shg
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Xbii
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Xbiii
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aab IV li I The cranial nerves develop in an anterior-to-posterior sequence, starting at 8 dpf aab III lap aab II aab IV The ramuli innervating the corresponding (30°C). muscles are first detectable when od IV aab I le I aab III od IV muscle fibers are starting to form (detected with an antibody against desmin, Fig.aab IV aab II cbi aab III 2). The trigeminal, or fifth, cranial nerve (V) shows a similar development as in Daaab I nio rerio (Raible & Kruse 2000) and different from all other osteognathostome taxa. The two H major ramii, the ramus ophthalmicus and ramus maxillomandibularis grow out of the brain as one nerve and enter one common ganglion (Fig. 2 A,B) from earD ly on. In other non-teleostean osteognathostomes, the ramus ophthalmicus and the maxillomandibularis develop as two distinct nerves withEyean associated gantvramus IV ov II in later developmental stages. The only other taxon glion andimaare fused secondarily ov I tv IV showing a similiar developmental pattern are sharks in which it may haveovevolved III ov III cbe tv IV ov II convergently. ov I
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Vimp
V+VIIih
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VIIhm
VIIhm
VIIhy VII hh
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Xlt
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Vop
Vop
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Vmx Vop
Vop
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Vmn
Vmx Vmn
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Vmx Vmn
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Vmx Vmn
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Vop
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Vop Vmx
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C`
A` B 10dpf
A 9dpf
Eye
A early 10dpf
A` B late 10dpf
B` C 12dpf
D`
A`B 10dpf
A 9dpf
Eye
Vmx
Vmn
C`
Vmn
lamprey r4
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Vop
Osteognathostomata
Vop Vmx
Vmn
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Eye
Eye
B late 10dpf
C 12dpf B`
Eye
B` C 11dpf
C` E 13dpf
Eye Eye
B 10dpf
D 12dpf C`
B`C 12dpf
D`
Abbreviations nerves: V, trigeminal nerve; Vam, trigeminus ramulus adductor mandibulae; Vdop, trigeminal ramulus dilatator operculi; Vima, trigeminal ramulus intermandibularis anterioris; Vimp, trigeminal ramulus intermandibularis posterioris; Vlap, trigeminal ramulus levator arcus platinus; Vman, trigeminal ramus mandibularis; Vmn, trigeminal ramus maxillomandibularis; Vmax, trigeminal ramus maxillaris; Vop, trigeminal ramus ophthalmicus; VII, facial nerve; VIIaX, communicating branchC`Dof12dpf the facial nerve to the vagal nerve; VIIah, facial ramulus D` C 12dpf E 13dpf C 11dpf C` C 12dpf D` adductor hyomandibulae; VIIhh, facial ramuli hyohyoidei; VIIhi, facial ramulus hyohyoideus inferioris; VIIhm, facial ramus hyomandibularis; VIIih, facial ramulus interhyoideus; VIIhy, facial ramus hyoideus; VIIop, facial ramulus ophthalmicus; VIIopm, facial ramulus of the dorsal opercular muscles; IX, glossopharyngeal nerve; X, vagal nerve; Xb2-Xb5, vagal ramulus branchialis 2 to 5; Xcb, vagal ramulus coracobranchialis; Xcbe, vagal ramulus coracobranchialis exterioris; Xcbi, vagal ramulus coracobranchialis interioris; Xcu, vagal ramulus cucullaris; Xin, vagal ramus internus; Xlt, vagal ramus lateralis; Xlpo, vagal ramulus levator posterior; Xpc, vagal ramulus pharyngocleithralis; S1-S4, spinal nerve 1 to 4; Shg, spinal hypoglossal nerve; Slp, spinal nerve D 12dpf D`E 13dpf C 12dpf D` E 13dpf 1 ramulus levator pectoralis; PFN, pectoral fin nerves.
r1+r2
Eye
Eye
Eye
Eye
Eye
Abbreviations muscles: aap, adductor arcus palatini; ah, adductor hyomandibulae; am, adductor mandibulae; aop, adductor operculi; bs, branchiostegal muscles; cbe, coracobranchialis externus; cbi, coracobranchialis internus; cu, cucullaris; dop, dilatator operculi; hi, hyohyoideus inferioris; hhs, hyohyoideus superioris; ih, interhyoideus; ima, intermandibularis anE 13dpf terioris; imp, intermandibularis posterioris; lap, levator arcus palatini; lop, levator operculi; lpe, levator pectoralis; pfm, pectoral fin muscles; ph, protractor hyoidei; sh, sternohyoideus. E 13dpf
r3
r1
r4
r2
r3
r4
Vop Vop Vmx Vmx
Vmn
Vmn
Gnathostomata
Eye
Abbreviations skeleton: BH, basihyal; CH, ceratohyal; HS, hyosymplectic; HH, hyohyal; IH, interhyal; MC, Meckel‘s Cartilage; NC, neurocranium; OC, otic capsule; PQ, palatoquadrate. References: Kuratani, S.; Nobusada, Y.; Saito, H.; Shigetani, Y. 2000. Morphological Characteristics of the Developing Cranial Nerves and Mesodermal Head Cavities in Sturgeon Embryos from Early Pharyngula to Late Larval Stages. zoological Science, 17, 911-933.; Raible, D. W.; Kruse, G. J. 2000. Organization of the Lateral Line System in Embryonic Zebrafish. The Journal Of Comparative Neurology, 421, 189-198.
Actinopterygii
Vmx
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Eye
Sarcopterygii
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The development of the head skeleton
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Eye
The development of the head muscles
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The trigeminal nerve (V) consists of three major ramii: the ophthalmic, the maxillary and the mandibular ramus (the latter two diverge from one main trunk, the maxillomandibular branch). In Sarcopterygii, basal Actinopterygii and the jawless lamprey the ophthalmic ramus develops as a separate nerve with a distinct ganglion fusing secondarily to the maxillomandibularis branch of the trigeminus. In sharks as well as N. furzeri (and Danio rerio in Raible et al. 2000) the ophthalmic nerve branches off from the maxillomandibularis ramus and is never observed as a separate nerve. Therefore it seems, that the developmental processes, leading to a relatively conserved pattern of the „adult“ trigeminal nerve might be more variable than previously thought. This raises questions about the origin(s) and homology of the opthalmic ramus of the trigeminus across jawed vertebrates. B late 10dpf
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The development of the branchiomeric cranial nerves lp
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VIIopm VIIhm VIIhy
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The trigeminal nerve one pattern and different processes VIIhm
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Vima
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BH V
C
Eye
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VIIop(cut) VIIaX V IX
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Vlap
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Vop
sh
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cu lpe a N. Figure 1: Visualization of methods. A, Optical section aop through lop ah with phosphotungstic furzeri larva at 16 dpf. Specimen was contrasted dop acid and scanned with synchrotron radiation at the DESY in Hamburg, lap Germany. The 3-axis view was produced using the AMIRA software. B, 3D reconstruction am of the optical sections in A. The reconstruction was done using the AMIRA software. Subsequently, colouring and image am was carried out using the MAYA software. C, Maximum inrendering tensity projection of a whole mount antibody staining of a 16 dpf larva from a lateral view. Muscles are stained with an antibody against desmin and are depicted in green. The cartilaginous elements of the cranium are stained with an antibody against collagen 2 and are depicted in red. D, Depth coding view of of a whole mount antibody staining of a 16 dpf larva from a lateral view. Structures in front of the am image are depicted in red whereas structures in the back merge into blue. Nerves are stained with an antibody against acetylated alpha-tubulin. the branchioima E, Schematic drawing of the main branches of cbi meric cranial nerves of the specimen bs shown in D. Scale bar is 200pfm µm.
Xlt
HS
S2
Figure 2: Development of the cranial nerves in N. furzeri. A-D, Depth-coding views of N. furzeri larvae at different developmental stages. Nerves are S4 stained with an antibody against acetylated alpha-tubulin. Specimens shown Eye S VII from a lateral view. A`-D`, Schematical drawings of the same specimens for X S1 Eye V S2 better illustration of fine nerve ramuli. F, MagnificationEyeof the branchial reS3 X S gion of the specimen shown in D. Red arrowheads indicate the dorsal branVII VII V X X X VII S4 V X V X X X chial ramuli of the glossopharyngeal (IX) and vagal (X) nerves innervating XX V the dorsal branchial muscles (levatores externi muscles). Blue arrowheads ov III S1 X V V VII VII VII (cut) S2 indicate the ramuliVIIinnervating the musculus levator internus 1 and 2. Orange arrowheads indicate the ramuli innervating the attrator arcus branchialium muscles. D 11dpf D` V VII V X S3 ov II VII X Green arrowheads indicate the venral ramuli of the IXth and XthIXcranial nerves innervating the ventral branchial musculature (musculi transversi ventrales , dilatator ov I IX Eye pharyngeus and coracobranchialis.S1Scale bar is 100 µm. Vop
IX
VIIop(cut) V
Slp
Vop
PR Slp S1
aop
OC
ah
aap
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Vmn
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NC
S3 Xb3 Xb2 Xb4
ima
VIIhiCHVIIhs
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VIIih
F32 E
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MC
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VIIaX
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VIIopm VIIhm VIIhy
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sh Vima
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lop
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VIIaX
Vmn Vmx
S3
Vlap
pfm
cbi cbe
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Vam
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Vmn Vmx
C`
Vman
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am
cbi cbe
S4
VIIaX
Vop
Xlt Xlt
Vmn Vmx
F
PR
Eye
imp
IX
aop
ah
ima
C
VIIopm IX
SC
Xlt
VIIop
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am
X
Vmx
IX
Vop
Yolk Sac
Shg
V VII
Vman
E
aop cu lop lpe
CH
MC
Vima
Xlt
S1 S2
Vima
VIII
Yolk Sac dop
Xlt
Xb3 Xb5 Xb4
sh
hs
VII
Xb
S3
PR X
VIIhy VIIhm
Eye
Vmax
bs
S2 S3
VII
SC
IX
Vmm
S2 S3
hi
Vmm
lap
S1
X
IX
S1
Xlt
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VIIaX
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ima
am X
Eye
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HB
Eye
B` am
Here we present the development of the cranial nerves, with special reference to the trigeminal nerve, in N. furzeri. Vman
VIIaX
VIIop
MB
S1
IX
VII
Vop
Vmax
Vop
BH HH ima
SC
Eye
Vmn Vmx
ah
dop
lap
Xbv Xpc
Vlap
BH
Vmn
A`
We used classical histological techniques (azan stained paraffin sections), clearing & staining as well as whole mount antibody staining combined with confocal laser scanning microscopy and 3D reconstruction of µCT data (see Fig. 1). Yolk Sac
Vam
IX
Vop
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cu
PQ
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MC
am
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lpe
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am
Eye
Vmm
Eye
CH
The cartilaginous skeleton, muscles and nerves of the head are completely differentiated at the time of hatching, BD around 13 days post-fertilization (dpf) at 30°C. r3
Xbii
A C
Xlt
S3
VII
V
Vop
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r2
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VIIopm
Vdop
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OC
HS
VIIaX
MB
dop
lap aap
To follow the development of the head in N. furzeri embryos and larvae we investigated the anatomy of the musculo-skeletal system of the head as well as the branchiomeric cranial nerves in young juveniles and adults as a „reference point“ for further morphological studies.
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Vertebrata
Figure 3: Cladogram of the major vertebrate taxa. For each taxon, the topography of the major branches of the trigeminal nerve (V) is shown in an embryonic (top) and the adult (below) condition. In the lamprey, Sarcopterygii and basal Actinopterygii, such as sturgeons, the opthalmic branch (Vop) develops separated from the maxillary (Vmx) and mandibular (Vmn) branch. In sharks and N. furzeri, the opthalmic branch develops from a common stem with the maxillomandibular branch. This might indicate a change in the developmental pattern of the trigeminal nerve in the evolutionary history of the teleost fishes compared to other Osteognathostomata. Scheme modified from Kuratani et al. 2000
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