Evolutionarily conserved morphogenetic movements at the vertebrate head–trunk interface coordinate the transport and assembly of hypopharyngeal structures

The vertebrate head–trunk interface (occipital region) has been heavily remodelled during evolution, and its development is still poorly understood. In extant jawed vertebrates, this region provides muscle precursors for the throat and tongue (hypopharyngeal/hypobranchial/hypoglossal muscle precursors, HMP) that take a stereotype path rostrally along the pharynx and are thought to reach their target sites via active migration. Yet, this projection pattern emerged in jawless vertebrates before the evolution of migratory muscle precursors. This suggests that a so far elusive, more basic transport mechanism must have existed and may still be traceable today. Here we show for the first time that all occipital tissues participate in well-conserved cell movements. These cell movements are spearheaded by the occipital lateral mesoderm and ectoderm that split into two streams. The rostrally directed stream projects along the floor of the pharynx and reaches as far rostrally as the floor of the mandibular arch and outflow tract of the heart. Notably, this stream leads and engulfs the later emerging HMP, neural crest cells and hypoglossal nerve. When we (i) attempted to redirect hypobranchial/hypoglossal muscle precursors towards various attractants, (ii) placed non-migratory muscle precursors into the occipital environment or (iii) molecularly or (iv) genetically rendered muscle precursors non-migratory, they still followed the trajectory set by the occipital lateral mesoderm and ectoderm. Thus, we have discovered evolutionarily conserved morphogenetic movements, driven by the occipital lateral mesoderm and ectoderm, that ensure cell transport and organ assembly at the head–trunk interface.


Tissue separation experiments
Tissue separation experiments were carried out in the occipital region of HH9-HH10 chicken embryos in ovo, implanting a 10µm thick piece of tantalum foil as impermeable barrier. To separate the occipital somites from the neural tube and notochord, a vertical incision was made along the wall of the neural tube, and the foil was pushed into the slit until it made contact with the endoderm (Suppl. Fig.2 C,D, Suppl. Fig.4a C,D). To separate the occipital somites from the lateral mesoderm, a vertical incision was made lateral to the somites, and the foil was inserted into the slit as before (Suppl. Fig.2 E,F, Suppl. Fig.4a E,F). To separate the somites from the overlying ectoderm, a shallow incision was made into the ectoderm lateral to the somite, a drop of dispase solution (Sigma) at 1mg/ml was applied using an aspirator, the ectoderm was peeled back, the slit was rinsed with PBS, and the foil was inserted to overlie the somites (Suppl. Fig.2 G,H, Suppl. Fig.4a G,H). The methodology is described in detail in Dietrich et al., 1997). Embryos were cultured for 36 hours to reach HH18-19.
Suppl. Fig.2b. Grafting of possible HMP attractants. Quail derived grafts are shown in brown, CellTracker Orange stained tissue culture cells in orange, protein loaded beads in blue and recipient areas in the host in turquoise.

(i) Implantation of rostrocaudally inverted occipital lateral mesoderm and ectoderm
Using flame-sharpened tungsten wire, the occipital lateral mesoderm and overlying ectoderm of the right side of HH9-10chicken embryos was excised in ovo.

Stage-matched quail donors were pinned down in a
Sylgard dish (Dow Corning), the left occipital lateral mesoderm and ectoderm were excised, aspired into a glass capillary to control its orientation, and released into the gap made in the host, dorsal (ectodermal) side up. This way, the rostrocaudal orientation of the graft was inverted while the original mediolateral orientation was maintained (Suppl. Fig.2b A,A'; Fig.9A).
In a similar fashion, lateral mesoderm and ectoderm from cervical, forelimb or flank levels was grafted (not shown). Embryos were harvested 36 hours after surgery at HH18-19.

(ii) Implantation of putative HMP attractants next to the caudal-most occipital somite
Using flame-sharpened tungsten needles, an insertion was made next to somites 4/5 of a HH10 chicken embryo in ovo. Pharyngeal endoderm of a pinneddown HH15-16 quail embryo was separated from surrounding tissues by applying a drop of 1mg/ml dispase (Sigma) with an aspirator, the tissue was transferred into the host with a serum-coated tip and manoeuvred into the slit (Suppl. Fig.2b B,B'; Fig.9B).
Embryos were harvested at stage HH20.