Relative quantification of zebrafish CHIA gene expression at ten stages of development. qPCR measurements of gene expression are shown from the 1 to 2 cells stage until 5 days post fertilization. The house keeping genes RNA polymerase II and 18S ribosomal subunit were used to normalize the measurements. According to quantitative gene expression levels, the CHIA genes can be roughly divided into two groups: (A) CHIA.1, CHIA.2, and CHIA.3 have very low levels of expression at the earliest developmental stages, but these rise sharply from day 3 to 4. (B) CHIA.4, CHIA.5 and CHIA.6 are more highly expressed at the earliest stages followed by a slower increase in expression. Interestingly, the CHIA.4 has an apparent maximum at the sphere stage and expression decreases at later stages, after the maternal to zygotic transition. Error bars indicate standard deviations.

CHIA gene expression from zygote to segmentation. (A–F) In situ hybridization results for the six earliest stages. In each panel the order is the same, top left: CHIA.1; top centre: CHIA.2; top right: CHIA.3; bottom left: CHIA.4; bottom centre: CHIA.5; bottom right: CHIA.6. The bar represents 1 mm in each case. (A–E) No differential pattern of gene expression can be distinguished in any of the initial stages of development. The early peak in CHIA.4 expression ( Fig. 3B) can be clearly seen in panels A and B. After sphere stage, all CHIA appear ubiquituously and lowly expressed (C–E). At 24 HPF the first distinguishable patterns can be seen (F). (G) Scale-up picture of the CHIA.4 ISH result, representative for patterns visible in all ISHs: the signal is concentrated in the developing eye (E) and brain (B), as well as at the myotome borders (MB).

CHIA expression patterns at pharyngula through to the early larval stages. (A–C) 2 DPF, (D–F) 3 DPF. The bar represents 1 mm except in F where it represents 0.4 mm. (A) 2 DPF panel of all CHIA (1–6) gene expression patterns. The lens of the eye and the caudal aorta seem to be the major tissues of expression of all the CHIA genes. The pectoral fin buds are visible in dorsal views of CHIA.2, CHIA.5 and CHIA.6 (arrowheads in A2, A5 and A6). (B) 2 DPF lateral close-up. The primary head sinus (PHS) can be seen as a curved line along the anteroposterior axis posterior to the eye. Extending dorsally immediately posterior to the tectum runs what appears to be the middle cerebral vein (MCV). Along the tail the caudal aorta (CA) can be seen extending all along the trunk and tail. (C) 2 DPF dorsal close-up. The lens (L) displays a strong signal at this stage. (D) 3 DPF panel of all CHIA gene expression patterns. (E) 3 DPF lateral close-up. The head blood vessels (HBV) can be seen at several places in the brain region. The tissue around the newly opened mouth (M), and the pharynx (P) and pharyngeal arches (PA) can be distinguished. The heart (H), caudal aorta (CA) and intestinal bulb (IB) also exhibit clear expression. (F) Dorsal close-up of the eye: the inner plexiform (IP) layer forms a clear cup shaped symmetrical pattern around the lens. Faint expression is visible in the optic nerve (ON) extending from the lens.

CHIA expression patterns at the larval stage; days 4 and 5. (A–D) 4 DPF larvae, (E) 5 DPF larvae. All bars represent 1 mm. (A) 4 DPF panel of all CHIA gene expression patterns. Expression in intestinal tissues become increasingly visible and is especially clear in the stomach for CHIA.1 (A1), and the liver for CHIA.5 and CHIA.6 (A5–6). (B) Lateral 4 DPF head close-up. The white matter (WM) can be distinguished clearly over the generally diffuse staining in the brain. (C) 6 μm section through the head of an embryo at 4 DPF. The white matter (WM) staining is outlined, and the pharyngeal arches (PA) and several head blood vessels (HBV) are also clearly discernable. Underneath is a schematic representation showing the approximate position of the slice in the embryo. (D) Dorsal 4 DPF head close-up. The habenulae (H) can be clearly distinguished in CHIA.4. (E) 5 DPF panel of all CHIA genes. CHIA.1, CHIA.2 and CHIA.3 (E1–3) are all nearly exclusively expressed in the lining of the stomach (S). CHIA.4, CHIA.5 and CHIA.6 much less pronounced so (E4–6). Some major blood vessels of the head are still visible such as the primordial midbrain channel (PMBC in E5). CHIA.6 seems to be primarily expressed in the liver (L in E6).