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. 2019 Jul 6:10:14.
doi: 10.1186/s13227-019-0128-3. eCollection 2019.

Gene profiling of head mesoderm in early zebrafish development: insights into the evolution of cranial mesoderm

Huijia Wang  1 , Peter W H Holland  2 , Tokiharu Takahashi  1
Affiliations

Gene profiling of head mesoderm in early zebrafish development: insights into the evolution of cranial mesoderm

Huijia Wang et al. Evodevo. .

Abstract

Background: The evolution of the head was one of the key events that marked the transition from invertebrates to vertebrates. With the emergence of structures such as eyes and jaws, vertebrates evolved an active and predatory life style and radiated into diversity of large-bodied animals. These organs are moved by cranial muscles that derive embryologically from head mesoderm. Compared with other embryonic components of the head, such as placodes and cranial neural crest cells, our understanding of cranial mesoderm is limited and is restricted to few species.

Results: Here, we report the expression patterns of key genes in zebrafish head mesoderm at very early developmental stages. Apart from a basic anterior-posterior axis marked by a combination of pitx2 and tbx1 expression, we find that most gene expression patterns are poorly conserved between zebrafish and chick, suggesting fewer developmental constraints imposed than in trunk mesoderm. Interestingly, the gene expression patterns clearly show the early establishment of medial-lateral compartmentalisation in zebrafish head mesoderm, comprising a wide medial zone flanked by two narrower strips.

Conclusions: In zebrafish head mesoderm, there is no clear molecular regionalisation along the anteroposterior axis as previously reported in chick embryos. In contrast, the medial-lateral regionalisation is formed at early developmental stages. These patterns correspond to the distinction between paraxial mesoderm and lateral plate mesoderm in the trunk, suggesting a common groundplan for patterning head and trunk mesoderm. By comparison of these expression patterns to that of amphioxus homologues, we argue for an evolutionary link between zebrafish head mesoderm and amphioxus anteriormost somites.

Keywords: Amphioxus; Anterior lateral plate mesoderm; Cardiopharyngeal field; Cranial lateral mesoderm; Cranial paraxial mesoderm; Head mesoderm; Head segmentation; Pharyngeal mesoderm; Zebrafish.

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Conflict of interest statement

Competing interestsThe authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Spatio-temporal expression of prechordal plate marker genes (gsc, pitx2 and isl1) during zebrafish early development. A–F Whole-mount in situ hybridisation of zebrafish prechordal plate/polster marker genes. Embryos were hybridised with gsc (Ai–vii, Bi–vii), pitx2 (Ci–vii, Di–vii) and isl1 probes (Ei–vii, Fi–vii). Expression of all three marker genes is detected in the pre-polster, a distinct group of cells located underneath the forebrain, at 75% epiboly stage (Ai, Ci, Ei). gsc and pitx2 transcripts are also detected in the posterior prechordal plate during early development (Ai–ii, Bi–ii, Ci–ii, Di–ii). From 7-somite stage onwards, expression of isl1 was also seen in the trigeminal placodes (Ev-vii, Fv-vii). White arrowheads indicate the expression of marker genes in the polster, the most anterior part of the prechordal plate. Brackets indicate strong expression of gsc (Ai, Bi) and graded expression of pitx2 from the anterior to the posterior tip in the prechordal plate during late gastrula period (Ci, Di). In all images, anterior is oriented to the top. Lateral images (B, D, F) are viewed from the left side of the embryos. 75E, 75% epiboly; hpf, hours post-fertilisation; ss, somite stage; TP, trigeminal placode. Scale bar: 100 μm
Fig. 2
Fig. 2
Spatio-temporal expression of cranial paraxial mesoderm marker genes (foxc1a and fsta) during zebrafish early development. A–F Whole-mount in situ hybridisation of zebrafish cranial paraxial mesoderm marker genes. The embryos were stained with foxc1a (Ai–vii, Bi–vii, Ci–vii) and fsta (Di–vii, Ei–vii, Fi–vii) probes. Both genes were expressed in the paraxial mesoderm, including the cranial paraxial mesoderm and the presomitic mesoderm. Black arrows indicate the cranial paraxial mesoderm. Anterior is to the top in dorsal and lateral views; in all transverse views, dorsal side is oriented to the top. Transverse views were taken at the level indicated by the dashed lines in lateral views. 75E, 75% epiboly; hpf, hours post-fertilisation; mb, midbrain; opv, optic vesicle; otv, otic vesicle; ss, somite stage. Scale bar: 100 μm
Fig. 3
Fig. 3
Spatio-temporal expression of cranial lateral mesoderm marker genes (tbx1, cyp26c1 and alx1) during zebrafish early development. A–I Whole-mount in situ hybridisation of zebrafish cranial lateral mesoderm marker genes. Embryos were hybridised with tbx1 (Ai–vii, Bi–vii, Ci–vii), cyp26c1 (Di–vii, Ei–vii, Fi–vii) and alx1 (Gi–vii, Hi–vii, Ii–vii) probes. Expression of all three genes in the cranial lateral mesoderm can be seen from bud- to 5-somite stage (Aii–iv, Dii–iv, Gii–iv, arrows). Black arrows indicate the cranial lateral mesoderm marked by the three genes. White arrowheads show the tbx1-expressing posterior region in the cranial paraxial mesoderm. Asterisks highlight the neural crest population labelled with alx1. In dorsal and lateral views, anterior is oriented to the top; in transverse views, dorsal side is oriented to the top. Transverse views were taken at the level indicated by the dashed lines in lateral views. 75E, 75% epiboly; d, diencephalon; hpf, hours post-fertilisation; otp, otic placode; r, rhombomere; ss, somite stage. Scale bar: 100 μm
Fig. 4
Fig. 4
Spatio-temporal expression of anterior lateral plate mesoderm (ALPM) marker tbx20 during zebrafish early development. A–C Whole-mount in situ hybridisation of zebrafish ALPM marker gene. Embryos were hybridised with tbx20 (Ai–vii, Bi–vii, Ci–vii) probes. tbx20 transcript can be first observed around bud stage in a reverse U-shaped domain (Aii). This domain covers the anterior lateral plate mesoderm. Transverse views (Ci–vii) were taken at the level shown by the dashed lines illustrated in lateral images (Bi–vii). Anterior is to the top in dorsal and lateral views. In all transverse views, dorsal side is oriented to the top. White arrowheads indicate the prospective heart primordium. 75E, 75% epiboly; hpf, hours post-fertilisation; ss, somite stage. Scale bar: 100 μm
Fig. 5
Fig. 5
Expression patterns of foxc1a, tbx1 and tbx20 genes resolve into three longitudinal strips in zebrafish head mesoderm (3ss, 11hpf). Confocal microscope images of triple in situ hybridisation of foxc1a (dark blue), tbx1 (red) and tbx20 (green) mRNA. Ai–iv Dorsal views of a whole-mount zebrafish embryo at 3ss (11hpf), anterior is oriented to the top. Note that the embryo was slightly tilted towards to the left when embedded in agarose gel to show better the separation of expression regions on the right side of the embryo. The innermost paraxial strip (dark blue) expresses foxc1a gene, then a more lateral strip (red) expresses tbx1, and finally, the most lateral strip (green) expresses tbx20. The boundaries of the three regions overlap. Bi–iv Cross sections of a zebrafish embryo at 3ss showing the right side of the head mesoderm, dorsal is oriented to the left-top. The level of sectioning is at the anterior hindbrain shown as the dotted line in Ai. Scale bar: 50 μm
Fig. 6
Fig. 6
Schematic illustration of zebrafish cranial mesoderm pattering at early development stage (3ss) in comparison with chick embryo (HH6). Dorsal view of a zebrafish embryo at 3ss and the rostral region of a chick embryo at HH6. By the end of gastrulation, a distinct group of cells form the polster which lies at most anterior part of the prechordal plate mesoderm. These cells are marked by the expression of pitx2 (yellow, left) in zebrafish but not in chick. Posterior to the polster, the zebrafish cranial mesoderm (left) resolves into three bilateral strips along the mediolateral axis. The innermost region is the cranial paraxial mesoderm (dark blue) located adjacent to the notochord and marked by the expressions of foxc1a and fsta; lateral to the paraxial mesoderm is the cranial lateral mesoderm (red) marked by the expression of tbx1, cyp26c1 and alx1 genes; the outermost strip is the anterior lateral plate mesoderm (ALPM, green) labelled with tbx20. Note that tbx1 gene is also expressed in the posterior region (*) of the cranial paraxial mesoderm. The boundaries between the three mediolateral strips partially overlap. In contrast to zebrafish embryos, chick head mesoderm lacks the intermediate strip (red) of cranial lateral mesoderm. Instead, the paraxial mesoderm marked by FoxC1 (dark blue) is further sub-regionalised into the anterior part marked by Pitx2 (yellow/blue stripes) and posterior (*) by Tbx1 (red/blue stripes). Chick Tbx20 (green) is expressed in both anterior and posterior lateral mesoderm at this stage
Fig. 7
Fig. 7
Comparison of the three-strip expression patterns of homologous genes between zebrafish and amphioxus embryos. Dorsal view of a zebrafish embryo at early segmentation period (3ss) and an amphioxus embryo at mid-neurula (7ss). In zebrafish head mesoderm at this developmental stage, expression patterns of marker genes resolve into three parallel bilateral strips. The innermost region marked by foxc1a (dark blue) expression continues to the posterior paraxial mesoderm which is segmented by somites. In the amphioxus embryo, FoxC is expressed in the somites all along the body. Lateral to the paraxial mesoderm in zebrafish is the cranial lateral mesoderm (red) marked by the expression of tbx1, cyp26c1 and alx1 genes. In the amphioxus embryo, Tbx1/10 expression spans paraxial (somites) and ventral mesoderm. The latter is known to be homologous to vertebrate lateral mesoderm (wisteria), and Tbx1/10 expression here is limited to the most anterior pharyngeal region. In the somites, Tbx1/10 is expressed only in the ventral half of all the somites shown as red/blue stripes. Alx gene shows a similar expression pattern except that its somite expression is limited to the anteriormost region. In amphioxus, all three Cyp26 genes are expressed only in the most anterior somites, but not in the ventral mesoderm. The outermost strip is the anterior lateral plate mesoderm (ALPM, green) labelled with tbx20. In the amphioxus embryo, Tbx20 is expressed in the ventral mesoderm from anterior to posterior region. Note that the prechordal plate is not shown in this diagram
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