Completing the set of h/E(spl) cyclic genes in zebrafish: her12 and her15 reveal novel modes of expression and contribute to the segmentation clock

Abstract

Somitogenesis is the key developmental process that lays down the framework for a metameric body in vertebrates. Somites are generated from the un-segmented presomitic mesoderm (PSM) by a pre-patterning process driven by a molecular oscillator termed the segmentation clock. The Delta-Notch intercellular signaling pathway and genes belonging to the hairy (h) and Enhancer of split (E(spl))-related (h/E(spl)) family of transcriptional repressors are conserved components of this oscillator. A subset of these genes, called cyclic genes, is characterized by oscillating mRNA expression that sweeps anteriorly like a wave through the embryonic PSM. Periodic transcriptional repression by H/E(spl) proteins is thought to provide a critical part of a negative feedback loop in the oscillatory process, but it is an open question how many cyclic h/E(spl) genes are involved in the somitogenesis clock in any species, and what distinct roles they might play. From a genome-wide search for h/E(spl) genes in the zebrafish, we previously estimated a total of five cyclic members. Here we report that one of these, the mHes5 homologue her15 actually exists as a very recently duplicated gene pair. We investigate the expression of this gene pair and analyse its regulation and activity in comparison to the paralogous her12 gene, and the other cyclic h/E(spl) genes in the zebrafish. The her15 gene pair and her12 display novel and distinct expression features, including a caudally restricted oscillatory domain and dynamic stripes of expression in the rostral PSM that occur at the future segmental borders. her15 expression stripes demarcate a unique two-segment interval in the rostral PSM. Mutant, morpholino, and inhibitor studies show that her12 and her15 expression in the PSM is regulated by Delta-Notch signaling in a complex manner, and is dependent on her7, but not her1 function. Morpholino-mediated her12 knockdown disrupts cyclic gene expression, indicating that it is a non-redundant core component of the segmentation clock. Over-expression of her12, her15 or her7 disrupts cyclic gene expression and somite border formation, and structure function analysis of Her7 indicates that DNA binding, but not Groucho-recruitment seems to be important in this process. Thus, the zebrafish has five functional cyclic h/E(spl) genes, which are expressed in a distinct spatial configuration. We propose that this creates a segmentation oscillator that varies in biochemical composition depending on position in the PSM.

The genetics and embryology of zebrafish metamerism

“…Knockdown of her12 perturbs the oscillating expression of her1, her7, and deltaC, indicating a nonredundant function for her12 within the segmentation clock. In contrast, inhibition of her15 alone or in combination with other genes did not lead to a segmentation defect (Shankaran et al, 2007).…”

“…her11 appears only to oscillate in the anterior half of the tail bud, while her12 and her15 oscillations are confined to the posterior half of the tail bud ( Fig. 3; Sieger et al, 2006;Shankaran et al, 2007). Control of oscillating gene expression differs between the anterior PSM and the rest of the tail bud.…”

“…Subsequent studies showed that her7, her11, her12, and her15 oscillate by performing double in situ hybridization with her1 and observing cyclical changes in expression in correlation with changes in her1 expression ( Fig. 3; Oates and Ho, 2002;Gajewski et al, 2003Gajewski et al, , 2006Sieger et al, 2004;Shankaran et al, 2007).…”

“…In fact, her15 has recently been reported to be expressed in the anlagen of alternating segments ( Fig. 3; Shankaran et al, 2007).…”

“…Loss of her1 in combination with loss of other genes can perturb all somites. References: a , b (Sawada et al, 2000), c (Holley et al, 2002), d (Henry et al, 2002), e (Oates and Ho, 2002), f , g (Sieger et al, 2004), h , i , j , k (Pasini et al, 2001), l (Pasini et al, 2004), m (Oates et al, 2005a), n (Shankaran et al, 2007), o (Kawamura et al, 2005b), and p (Winkler et al, 2003).…”