Tissue regulation of somitic colloid-like1 gene expression
Highlights
► Somitic colloid-like1 gene expression is restricted to the 10 most rostral somites. ► A neural tube signal maintains somitic colloid-like1 gene expression. ► Colloid-like1 as a member of the molecular machinery operating in chick neck muscle formation.
Introduction
Somites are on the basis of the segmental pattern of the adult vertebrate body and give rise to vertebrae, intervertebral discs, ribs, the dermis of the back and all skeletal muscles of the adult body. Somites form along the antero-posterior embryonic axis, bilaterally to the neural tube and notochord, budding off from the cranial end of the presomitic mesoderm (PSM) [1] while new cells are caudally added as a consequence of gastrulation [2]. Initially, the somite is formed by a sphere of epithelial cells but, as development proceeds, its ventral part undergoes an epithelial-to-mesenchymal transition, forming the sclerotome. Meanwhile, its dorsal part remains epithelial and constitutes the dermomyotome, which later originates dermatome and myotome. In the latter, two domains can be defined from which the two sets of the body skeletal muscles arise; a dorsomedial/epaxial and a ventrolateral/hypaxial. Signals from surface ectoderm (SE), neural tube, notochord and lateral plate mesoderm (LPM) have been identified as crucial for somitic axes specification [3]. Bone Morphogenetic Protein 4 (BMP4) is produced by both dorsal neural tube and LPM and was shown to be crucial in several aspects of somite differentiation [4]. Several studies in Xenopus, chick and mouse revealed that secreted proteins like Noggin and Chordin create a gradient of BMP4 by directly binding to it, thus preventing an interaction with its receptor [5]. A further level of regulation is introduced by the secreted zinc metalloprotease, Tolloid, which belongs to the conserved family BMP1/Tolloid-like [6]. In early embryo, these proteins have been described as positive regulators of BMP4 activity by proteolytically cleaving Chordin, generating small fragments and thus reducing its affinity to BMP4 [7]. Contrarily to other species, very little is known about the function of these metalloproteases in the chick. Two BMP1/Tolloid family members have already been cloned, chicken BMP1/Tolloid and colloid-like1, and their expression patterns described (bmp1/Tolloid [8], [9]; colloid-like1 [10]). In this work, we characterise somitic colloid-like1 gene expression in embryos up to stage 15+HH. We assess the in vivo regulation of this expression by the different surrounding embryonic tissues such as the axial structures, neural tube and notochord, SE and flanking LPM. The data obtained allows us to propose a function of somitic colloid-like1 gene expression in neck muscle formation.
Section snippets
Chicken embryos
Fertilised chicken (Gallus gallus domesticus) eggs were obtained from commercial sources, stored at 16 °C and incubated in a 45% humidified atmosphere at 37 °C. Incubated embryos were harvested, washed in PBS without Ca2+/Mg2+ and staged according to Hamburger and Hamilton classification system [11]. Embryos of stages 7–15+HH were fixed overnight at 4 °C in a 4% formaldehyde solution and then progressively dehydrated in methanol series and stored in 100% methanol at −20 °C.
RNA probes
Colloid-like1, BMP4,
In ovo surgical slit
A small window was made in the eggshell and an Indian ink:PBS (1:1) solution was injected into the sub-blastodermic cavity. In embryos ranging from 10HH to 12HH, the right row of somites was separated from axial organs by a slit made through the three germ layers (Fig. 2A), along four to six somites posteriorly to the second to fourth most cranial somites. Operated embryos were reincubated for 12–16 h, harvested, fixed, dehydrated as described above and kept in methanol at −20 °C for in situ
Somitic colloid-like1 gene expression is restricted to the 10 most rostral somites
We evaluated the somitic expression of colloid-like1 gene by whole-mount in situ hybridisation and section analysis of embryos ranging from stages 6HH [11] to 15HH (Fig. 1). In embryos from stages 7+HH to 15+HH, colloid-like1 transcripts are broadly detected in all epithelial somites since they bud off from PSM (Fig. 1A, A2, B, C, C1, and C2). Surprisingly, from stage 10HH onwards, no colloid-like1 expression can be observed in forming epithelial somites, with somitic expression always
Acknowledgments
The authors would like to thank Sólveig Thorsteinsdóttir for her helpful ideas and suggestions and Joaquín Rodriguez-Léon for sending us the Wnt inhibitor. This work is supported by national Portuguese funding through FCT - Fundação para a Ciência e a Tecnologia, project ref. PEst-OE/EQB/LA0023/2011 and Centre for Molecular and Structural Biomedicine, CBME/IBB, LA.Tomas Pais de Azevedo is funded by PTDC/SAU-OBD/099 758/2008 and Lisa Gonçalves is funded by SFRH/BPD/65652/2009.
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These authors contributed equally for this work.