Folian-cv1 Is a Member of a Highly Acidic Phosphoprotein Class Derived From the Foliated Layer of the Eastern Oyster (\u3ci\u3eCrassostrea virginica\u3c/i\u3e) Shell and Identified in Hemocytes and Mantle

The proteins derived from the foliated shell layer of the oyster, Crassostrea virginica, are unusually acidic and highly phosphorylated. Here we report the identification of a gene encoding a member of this class of phosphoproteins that we collectively refer to as folian. Using an in silico approach, a virtual probe was constructed from an N-terminal sequence (DEADAGD) determined for a 48 kDa folian phosphoprotein and used to screen an oyster EST databank. A sequence that matched the N-terminus of the 48 kDa protein was found and used to identify the full-length gene from a C. virginica BAC library. The molecular weight of the deduced gene product is 32 kDa and was named folian-cv1. Genomic Southern analysis revealed two variants of the gene. The mature protein is composed of 43.3% Asp, 32.6% Ser, and 9.1% Glu with 37.5% of the amino acids of the protein potentially phosphorylated. The primary sequence of folian-cv1 is organized in blocks, with a short relatively hydrophobic block at the N-terminus and with the remainder containing low complexity regions largely dominated by aspartic acid and serine. Overall, the protein is predicted to be highly disordered. PCR and sequence analyses identified folian-cv1 expression in the mantle and hemocytes. Immuno-histochemical staining of mantle tissue reveals that cells of the shell-facing epithelium and in the periostracal groove secrete a continuous layer of folian-positive material and that folian-positive hemocytes move through the mantle epithelium. The function in shell formation of folian proteins including folian-cv1 is not known. However, based on the complexity of this class of proteins and the two methods of their delivery to the region of shell formation, it is possible they are involved in diverse ways in this process

<i>IRX1</i> and <i>IRX2</i> are misexpressed in Araucana<i>^Rp</i> tailbud.

<p><i>IRX1</i> expression pattern (A–F,M–P). (A,C,E) Control embryos express <i>IRX1</i> in the neural tube. <i>IRX1</i> expression in controls matches Araucana<i>^Rp</i> at HH14 (A,B), but is misexpressed in Araucana<i>^Rp</i> tailbud at HH15 (D-arrowhead). Normal expression at level of somites is within neural tube (transverse section, M). Misexpression in Araucana<i>^Rp</i> can be seen at the level of the chordoneural hinge (transverse section-N and sagittal section-P) compared to expression in HH15 control (sagittal section-O). Misexpression is maintained through HH16 in Araucana<i>^Rp</i> (F-arrow) as compared to control (E). <i>IRX2</i> expression pattern (G–L,Q). (G,I,K) Control embryos do not express <i>IRX2</i> in the tailbud. No difference in expression between controls and Araucana<i>^Rp</i> seen at HH14 (G,H). <i>IRX2</i> is misexpressed in HH15 (J-arrowhead) and HH16 (L-arrowhead) Araucana<i>^Rp</i>. Transverse section of <i>IRX2</i> (Q) shows expression similar to <i>IRX1</i> at level of chordoneural hinge. A–L - anterior to top. M,N,Q - dorsal to top. O,P - dorsal to left, anterior to top. Abbreviations: nt-neural tube, s-somite, n-notochord.</p

Skeletal analysis shows Araucana<i>^Rp</i> lack caudal vertebrae.

<p>Adult Araucana<i>^Rp</i> male and female birds shown in a composite image (A) (courtesy of Fritz Ludwig). Note the characteristic rounded rump, lacking tail structures. Skeletons of control (B) and Araucana<i>^Rp</i> (C) birds (courtesy of the ACA). The free vertebrae and pygostyle are missing in the Araucana<i>^Rp</i> skeleton (arrow). E18 embryos stained with Alcian Blue in control (D) and Araucana<i>^Rp</i> (E). Araucana<i>^Rp</i> embryos lack the free vertebrae and pygostyle. Arrowheads indicate lateral processes. Vertebral elements are numbered from the first free vertebrae (1–5). The more posterior vertebral elements (6–11) fuse to form the mature pygostyle after hatching. FV-free caudal vertebrae, P-pygostyle, S-sacral vertebrae.</p

Araucana<i>^Rp</i> form fewer somites.

<p><i>MESO1</i> expression in control (A,C) and Araucana<i>^Rp</i> (B,D) embryos at HH18-19. Note downregulation of <i>MESO1</i> in HH19 Araucana<i>^Rp</i> (D-arrowhead) compared to controls (C-arrowhead). <i>DACT2</i> expression in control (E,G) and Araucana<i>^Rp</i> (F,H) from HH19-20. Inset in G and H are sagittal sections through the paraxial mesoderm from respective embryo. Note decrease in somites labeled by <i>DACT2</i> at HH20 in Araucana<i>^Rp</i> (H-arrowhead) compared to control (H-arrowhead). Anterior up in whole mount and section insets. (I) Graph showing number of somites compared to embryonic stage (HH stages 16–25). Araucana<i>^Rp</i> have significantly fewer somites beginning at HH20 (T-test, asterisk marks p<0.01) than controls. Control-Black, Araucana<i>^Rp</i>-Grey.</p

Role of proliferation and apoptosis in Araucana<i>^Rp</i> tailbud development.

<p>EdU labeled proliferating cells in tailbud sagittal sections at HH15-20 in controls. (A,C,E,G) and Araucana<i>^Rp</i> (B,D,F,H) embryo tailbud. Green is EdU labeled proliferating cells, red is TO-PRO-3 Iodide labeled nuclei. Arrows denote decreased regions of proliferation in Araucana<i>^Rp</i> compared to controls. (I) Quantification of all TBM cells for control and Araucana<i>^Rp</i> labeled with TO-PRO-3 Iodide. (J) Quantification of proliferating TBM cells for control and Araucana<i>^Rp</i> labeled with EdU. TUNEL labeled apoptotic cells in tailbud sagittal sections from HH stages 15–20 of controls. (K,M,O,Q) and Araucana<i>^Rp</i> (L,N,P,R) embryo tailbud. Red is TUNEL labeled apoptotic cells, blue is Hoechst labeled nuclei. Arrows denote increased areas of apoptosis in Araucana<i>^Rp</i> compared to controls. (S) Quantification of apoptotic TUNEL labeled cells in control and Araucana<i>^Rp</i> TBM. Araucana<i>^Rp</i> have significantly more TUNEL positive cells beginning at HH17 than controls. T-test, asterisk marks p<0.05. Control-Black, Araucana<i>^Rp</i>-Grey. Same embryo (two different sections) was used for both TUNEL and EdU labeling. Anterior is towards the top, dorsal is towards the left in all sections.</p

Location of small variants associated with rumplessness in the 740 kb critical region.

<p>Top: positional map of the 740 kb critical region located on <i>Gallus gallus</i> chromosome 2 (GGA2). Location of <i>IRX1</i> and <i>IRX2</i> is indicated, with numbers of small variants (SV) (SNPs, insertions and deletions) and their general location and frequency indicated. No small variants were found within <i>IRX1</i> or <i>IRX2</i> coding sequence. Bottom: relative position of SNPs used to build new critical interval within the established Araucana<i>^Rp</i> haplotype block. Rumpless Araucana<i>^Rp</i> haplotype in blue, with associated SNPs, haplotype from tailed Araucana in yellow. New critical interval defined by RB1 tailed Araucana highlighted by dashed lines.</p

A Novel Gain-Of-Function Mutation of the Proneural <i>IRX1</i> and <i>IRX2</i> Genes Disrupts Axis Elongation in the Araucana Rumpless Chicken

<div><p>Axis elongation of the vertebrate embryo involves the generation of cell lineages from posterior progenitor populations. We investigated the molecular mechanism governing axis elongation in vertebrates using the Araucana rumpless chicken. Araucana embryos exhibit a defect in axis elongation, failing to form the terminal somites and concomitant free caudal vertebrae, pygostyle, and associated tissues of the tail. Through whole genome sequencing of six Araucana we have identified a critical 130 kb region, containing two candidate causative SNPs. Both SNPs are proximal to the <i>IRX1</i> and <i>IRX2</i> genes, which are required for neural specification. We show that <i>IRX1</i> and <i>IRX2</i> are both misexpressed within the bipotential chordoneural hinge progenitor population of Araucana embryos. Expression analysis of <i>BRA</i> and <i>TBX6,</i> required for specification of mesoderm, shows that both are downregulated, whereas <i>SOX2</i>, required for neural patterning, is expressed in ectopic epithelial tissue. Finally, we show downregulation of genes required for the protection and maintenance of the tailbud progenitor population from the effects of retinoic acid. Our results support a model where the disruption in balance of mesoderm and neural fate results in early depletion of the progenitor population as excess neural tissue forms at the expense of mesoderm, leading to too few mesoderm cells to form the terminal somites. Together this cascade of events leads to axis truncation.</p></div

ISH expression pattern of <i>RALDH2</i>, <i>CYP26A1</i>, <i>WNT3A</i> and <i>FGF8</i> during tail development.

<p><i>RALDH2</i> expression in control (A) and Araucana<i>^Rp</i> (B) embryos at HH18. Note in Araucana<i>^Rp</i> the truncated tail at HH18 coupled with the close expression of <i>RALDH2</i> in the formed somites. Posterior-most expression of <i>RALDH2</i> is marked with arrowhead. <i>CYP26A1</i> expression in control (C) and Araucana<i>^Rp</i> (D) embryos at HH16. Note the lack of expression in Araucana<i>^Rp</i> tailbud (arrowhead). <i>WNT3A</i> expression in control (E,G) and Araucana<i>^Rp</i> (F, H) embryos from HH17-18. Note lack of expression in tailbud mesenchyme in Araucana<i>^Rp</i> (arrowheads). <i>FGF8</i> expression in control (I,K) and Araucana<i>^Rp</i> (J,L) embryos from HH stages 17–18. Note the downregulation of expression in tailbud beginning at HH17 in Araucana<i>^Rp</i> (arrowheads). Insets are transverse sections at level of tailbud. Anterior is top in whole mount images. Dorsal is top in transverse section insets. Sections were taken at approximate level of black bars.</p