Seb4 is expressed in mesodermal and ectodermal derivatives

Analysis of the spatial and temporal in situ expression pattern of a factor can shed light on its biological function in many ways. For example overlapping expression domains can give information about potential interaction partners, or upstream/downstream regulation.

Seb4 was shown to be a direct target of MyoD (see 1.3.4; (Jasper, 1998)), so, first of all the RNA expression pattern of both factors were compared by RNA in situ hybridization (ISH; Figure 13A).

In blastula stage embryos (st. 9), zygotic myoD expression is not significantly induced yet, but a basal, maternal, ubiquitous myoD expression exists. The same is true for seb4, low, maternal RNA expression is detectable by RT-PCR (Fetka et al, 2000), but not by ISH yet. The myoD expression is induced in prospective myoblasts in the gastrula stage (st. 11) in an omega-shaped pattern around the blastopore, excluding the organizer region above the dorsal lip. Zygotic seb4 is induced in two small regions within the myoD expression domain flanking the dorsal lip/organizer. Neurula stage embryos (st. 18) show an identical myoD and seb4 expression pattern in the unsegmented, paraxial mesoderm consisting of presumptive myoblasts. In tailbud stage embryos (st. 30), myoD is expressed in the myotome and begins to be down-regulated in postmitotic, differentiating myocytes. Seb4 is likewise expressed in the myocytes of the myotome, but also in the myocardium and in ectodermal derivatives like the lens, the ventral part of the otic vesicle, some cranial placodes and developing lateral line primordia (Schlosser & Northcutt, 2000).

In the next experiment, the developmental Seb4 expression was followed in situ on the RNA level (ISH; 1-8) as well as on the protein level by immunocytochemistry (ICC; 1’-8’) in comparable embryonic stages (Figure 13B). In the early blastula (st. 7; Figure 13B1) the maternal RNA contribution of seb4 is located in the animal hemisphere. Seb4 protein is also localized in low amounts in the animal half of an early blastula embryo (B1’).

In the gastrula stage embryo (B2), zygotic seb4 transcription is induced in two small domains flanking the organizer region around the blastopore.

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Figure 13: In situ expression pattern of Seb4 in Xenopus laevis

embryos analysing the RNA and protein level. (A) Seb4 and myoD RNA in situ

hybridization in blastula, gastrula, neurula and tailbud stages. (B) Comparative analysis between Seb4 RNA (1-8) and protein (1’-8’) expression/localization in several stages by ISH (1-8) and ICC (1’-8’), respectively. Panels B1, 2’, 4-8’ lateral view; 1’ animal view; 2 posterior view; 3, 3’ dorsal view. Inlay in panel 3: crosssection perpendicular to A-P axis as indicated by white dashed line. Arrows in 5, 5’ point at the retina and lens, respectively. White squares in 7, 7’ are shown as enlargements in 8, 8’.

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At this stage the protein is still distributed over the animal half; no distinct protein domains are visible yet (B2’).

In neurula stage embryos (B3, 3’) the RNA and protein expression domains are located in the paraxial mesoderm and correlate with each other. In early tailbud stages, seb4 RNA expression is induced in the retina and in the otic vesicle (auditory vesicle; ear), whereas Seb4 protein cannot be detected yet in these ectodermal derivatives (B4, 4’).

Seb4 expression in the myoblasts correlates on the RNA and protein level. In later tailbud stages (B5, 5’) seb4 mRNA is expressed strongly in the retina (arrow) as well as in the myocardium. Seb4 protein is now also found in the eye, where it is expressed in the lens (arrow) and absent from the retina. The same is true for the proctodeum, where Seb4 protein is present, but Seb4 mRNA cannot be detected. Heart muscle and skeletal muscle in the somites show both Seb4 RNA and protein expression. In even later stages, after 2d of development (st. 34; 6 and 6’), the expression pattern of seb4 RNA and protein correlate in all tissues: striated muscle, heart muscle, lens and otic vesicle. Seb4 protein that was earlier (and later; see below) found in the anal region, has disappeared at stage 34. After 3d (st. 42; 7 and 7’) seb4 RNA expression begins to be down-regulated in the dorsal myocytes, whereas the protein is still detectable. At this time Seb4 is strongly expressed in the differentiated ventral body wall muscle. The ventral muscles have migrated ventrally and retain segmental identity, by the lack of fusion between the adjacent myotubules (Martin & Harland, 2001). Here, Seb4 protein appeared again in the anal region, whereby Seb4 mRNA seems absent.

Seb4 expression is also induced in the head in some cranial and facial placodes, in cells around the eye belonging the developing lateral line primordia, epibranchial placodes and trigeminal placodes (Schlosser & Northcutt, 2000). Most strikingly, seb4 RNA expression in the eye is still restricted to the lens, whereas Seb4 protein is clearly localized to the retina, and excluded from the lens (8 and 8’).

Altogether, Seb4 is expressed in a distinct tissue specific pattern in mesodermal and ectodermal derivatives. Including the neurula stages, its mesodermal expression domain mainly overlaps with the expression domain of myoD. At tailbud stages Seb4 is expressed not only in the somites like MyoD but is also found in the lens, the ear, and the heart. Seb4 expression correlates on the RNA and protein level in the myoblasts. Exceptions to this rule occur in the anal region and during eye differentiation in early tailbud and late tadpole stages.

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