Spatio-temporal expression of prechordal plate marker genes (gsc, pitx2 and isl1) during zebrafish early development. AF 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

 

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Stage Range: 75%-epiboly to 10-13 somites

Spatio-temporal expression of anterior lateral plate mesoderm (ALPM) marker tbx20 during zebrafish early development. AC Whole-mount in situ hybridisation of zebrafish ALPM marker gene. Embryos were hybridised with tbx20 (Ai–vii, Bi–vii, Ci–vii) probes. tbx20transcript 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

 

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

 

Spatio-temporal expression of cranial paraxial mesoderm marker genes (foxc1a and fsta) during zebrafish early development. AF 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

 

Spatio-temporal expression of cranial lateral mesoderm marker genes (tbx1, cyp26c1 and alx1) during zebrafish early development. AIWhole-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

 

Acknowledgments
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