FishFace

By 24 hpf, neural crest cells have populated the two anterior pharyngeal arches (pa1, pa2). The anterior region of pharyngeal arch 1 surrounds the oral ectoderm (oe), which is GFP-negative in fli1a:EGFP transgenic fish. Cells positioned ventral to the oral ectoderm in pharyngeal arch 1 contribute to Meckel’s cartilage and associated bones, while those positioned dorsally and posteriorly contribute to the palatoquadrate and portions of the neurocranium, along with their associated bones. Cells positioned ventrally in the second pharyngeal arch contribute to the ceratohyal and associated bones, while those positioned dorsally and posteriorly contribute to the hyosymplectic and associated bones. The first pharyngeal pouch (pp1), which is GFP-negative in fli1a:EGFP transgenic fish, dorsally separates pharyngeal arches 1 and 2, which contact each other ventrally.

By 28 hpf, pharyngeal arches 1 and 2 (pa1, pa2) have begun to converge (narrow) along the anterior-posterior axis and extend (lengthen) along the dorsal-ventral axis compared with the dimensions of these axes at 24 hpf. The contact between ventral cells of pharyngeal arches 1 and 2 grows as the arches extend along the dorsal-ventral axis. Pharyngeal pouch 1 (pp1), which is GFP-negative in fli1a:EGFP transgenic fish, maintains the dorsal separation between pharyngeal arches 1 and 2. The oral ectoderm (oe), which also is GFP-negative in fli1a:EGFP transgenic fish, continues to separate dorsal and ventral cells in the anterior region of pharyngeal arch 1.

By 32 hpf, pharyngeal arches 1 and 2 (pa1, pa2) continue to extend (lengthen) along the dorsal-ventral axis. Pharyngeal pouch 1 (pp1), which is GFP-negative in fli1a:EGFP transgenic fish, maintains the dorsal separation between pharyngeal arches 1 and 2, while the oral ectoderm (oe), which also is GFP-negative in fli1a:EGFP transgenic fish, continues to separate dorsal and ventral cells in the anterior region of pharyngeal arch 1. Pharyngeal arch 1 has expanded further anteriorly along both dorsal and ventral surfaces of the oral ectoderm, compared to that seen at 28 hpf. Also, some neural crest cells abutting pharyngeal pouch 1 appear to be aligning with the endodermal surface.

By 36 hpf, pharyngeal arch 1 (pa1) appears much longer than pharyngeal arch 2 (pa2) along the dorsal-ventral axis. Pharyngeal pouch 1 (pp1), which is GFP-negative in fli1a:EGFP transgenic fish, continues to separate pharyngeal arches 1 and 2 dorsally, while the oral ectoderm (oe), which also is GFP-negative in fli1a:EGFP transgenic fish, continues to separate dorsal and ventral cells in the anterior region of pharyngeal arch 1. Neural crest cells in the posterodorsal region of pharyngeal arch 1 and in the anterodorsal region of pharyngeal arch 2 appear to be aligned with the surface of pharyngeal pouch 1. A ventral view demonstrates that the ventral region of pharyngeal arch 1 has begun to extend medially.

By 42 hpf, pharyngeal arches 1 and 2 (pa1, pa2) have converged onto each other along the anterior-posterior axis and extended along the dorsal-ventral axis. Pharyngeal pouch 1, which is GFP-negative in fli1a:EGFP transgenic fish, appears to be regressing at this timepoint, although the neural crest cells maintain a tightly ordered arrangement in the dorsal region between pharyngeal arches 1 and 2. Pharyngeal arch 1 has increased in mediolateral thickness, and the oral ectoderm (oe), which is GFP-negative in fli1a:EGFP transgenic fish, is more constricted medially, from that seen at 36 hpf. The ventral region of pharyngeal arch 1 has further expanded medially, and the ventral region of pharyngeal arch 2 has begun to expand medially, compared to that seen at 36 hpf.

By 48 hpf, the ventral portions of pharyngeal arches 1 and 2 (pa1, pa2) have no clear boundary between them, and the dorsal portion of pharyngeal arch 1 is no longer visible from lateral view. The posterior pharyngeal arches 3-6 (pa3-6), which contribute cells to the ceratobranchials of the pharyngeal skeleton, are visible in a series with similar morphology. The ventral region of pharyngeal arch 1 has expanded extensively from that seen at 42 hpf, so that cells from the left and right sides of the embryo meet at the midline. In addition, the oral ectoderm (oe), which is GFP-negative in fli1a:EGFP transgenic fish, is more constricted medially. Compared to that seen at 42hpf, the ventral region of pharyngeal arch 2 has extended further medially, but cells of the left and right sides have yet to meet at the midline.

By 60 hpf, neural crest-derived cells have begun to condense into distinct skeletal components of the craniofacial apparatus (see Pharyngeal arch development--Skeletal elements). In fact, Alcian blue staining of cartilage matrix is visible in skeletal elements of pharyngeal arches 1 and 2 at this timepoint (data not shown). Cells of Meckel’s cartilage (Mk) and the palatoquadrate (pq) can be discerned in pharyngeal arch 1, while cells of the hyosymplectic (hs) and the ceratohyal (ch) are visible in pharyngeal arch 2. The ventral region of pharyngeal arch 2 has expanded extensively from that seen at 48 hpf, so that cells from the left and right sides of the embryo meet at the midline between the developing ceratohyals.