Czarkwiani A et al. (2013), Expression of skeletogenic genes during arm reg...

ECB-ART-43048

Gene Expr Patterns 2013 Dec 01;138:464-72. doi: 10.1016/j.gep.2013.09.002.

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Expression of skeletogenic genes during arm regeneration in the brittle star Amphiura filiformis.

Czarkwiani A , Dylus DV , Oliveri P .

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The brittle star Amphiura filiformis, which regenerates its arms post autotomy, is emerging as a useful model for studying the molecular underpinnings of regeneration, aided by the recent availability of some molecular resources. During regeneration a blastema initially is formed distally to the amputation site, and then a rapid rebuild is obtained by adding metameric units, which will eventually differentiate and become fully functional. In this work we first characterize the developmental process of the regenerating arms using two differentiation markers for muscle and skeletal structures - Afi-trop-1 and Afi-αcoll. Both genes are not expressed in the blastema and newly added undifferentiated metameric units. Their expression at different regenerating stages shows an early segregation of muscle and skeletal cells during the regenerating process, long before the metameric units become functional. We then studied the expression of a set of genes orthologous of the sea urchin transcription factors involved in the development of skeletal and non-skeletal mesoderm: Afi-ets1/2, Afi-alx1, Afi-tbr, Afi-foxB and Afi-gataC. We found that Afi-ets1/2, Afi-alx1, Afi-foxB and Afi-gataC are all expressed at the blastemal stage. As regeneration progresses those genes are expressed in a similar small undifferentiated domain beneath the distal growth cap, while in more advanced metameric units they become restricted to different skeletal domains. Afi-foxB becomes expressed in non-skeletal structures. This suggests that they might play a combinatorial role only in the early cell specification process and that subsequently they function independently in the differentiation of different structures. Afi-tbr is not present in the adult arm tissue at any stage of regeneration. In situ hybridization results have been confirmed with a new strategy for quantitative PCR (QPCR), using a subdivision of the three stages of regeneration into proximal (differentiated) and distal (undifferentiated) arm segments.

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Species referenced: Echinodermata
Genes referenced: alx1 ets1 foxb1 gata3 LOC100887844 LOC100893907 LOC115919910 LOC581907 LOC583082 LOC753216 tbr1

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	Fig. 1. (a–d) Schematic diagram of muscle and skeletal structures within the brittle star arm. (a) Oral view showing the spines, podia, and lateral and oral arm shields. (b) Aboral view showing spines, and lateral and aboral arm shields. (c) View of internal structures including the vertebrae and the intervertebral muscles. (d) Phalloidin staining revealing muscle structures in the proximal part of the 95% regenerating arm (aboral view). (e, f) Double fluorescent in situ hybridization showing Afi-trop1 localized to the podia (green arrows) at the oral side and intervertebral muscles (yellow arrow) at the aboral side of the 50% (e) and 95% (f) regenerating arm. Afi-αcoll is restricted to lateral arm shields and the base of the spines. The two differentiation markers are not co-expressed and neither is found at the distal tip (blue arrow) of the regenerating arm which confirms their use as markers for specified structures. (g,h) Chromogenic single in situ hybridization clearly showing expression in 95% regenerating arms of Afi-trop1 in the intervertebral muscles (yellow arrows) as well as the podia and Afi-αcoll in the lateral shields and base of the spines. Scale bars – 100 μm. Green – muscle structures, red – skeletal structures, 1 – oral arm shield, 2 – lateral arm shield, 3 – spines, 4 – aboral arm shields, 5 – vertebrae, A – podia, B – intervertebral muscles. Green arrows – podia, yellow arrows – intervertebral muscles, blue arrows – distal tip. OV – oral view, LV – lateral view. P – proximal, D – distal.
	Fig. 2. Structure of the blastema and expression of transcription factors. (A) Formation of metameric units and the specification of the blastema. At the very early regenerative phase the blastema is composed of two layers – an epithelium and the mass of proliferative and unspecified cells, of a total 50–100 cells. As the blastema elongates the proliferative area is becoming restricted to the distal-most tip of the newly forming arm and the blastema is organized into a three-layered structure, formed by the radial water canal, the midlayer and the epithelium. When metameric units defined by the appearance of pronounced podia and spines are being formed at the proximal end of the regenerate, the unspecified proliferative cells are only localized to the distal-most tip forming a growth zone that remains undifferentiated until the end of regeneration. (B) WMISH revealing differential expression patterns of transcription factors Afi-alx1, Afi-ets1/2, Afi-gataC, Afi-tbr and Afi-foxB in the blastema. All genes except for Afi-tbr are expressed in the blastema.Afi-foxB is localized to the epithelium, Afi-ets1/2 is ubiquitous and Afi-alx1 and Afi-gataC are both expressed within the thick midlayer of the blastema but not the distal-most tip. Scale bar: 100 μm, P – proximal, D – distal. White dashed lines – radial water canal.
	Fig. 3. Spatial expression of transcription factors Afi-alx1, Afi-ets1/2, Afi-foxB, Afi-tbr and Afi-gataC in the regenerating arm. Afi-tbr is not expressed at all in the regenerating arm. All four remaining genes are strongly upregulated directly beneath the undifferentiated distal cap. Proximal expression of Afi-alx1 is localized to the bases of the spines and podia and Afi-gataC only to the base of the spines, both at the 50% and 95% differentiation stages. Afi-ets1/2 shows a repetitive pattern of expression restricted to the site of developing vertebrae in the proximal regions of the regenerating arm. Expression of Afi-foxB is localized to the non-skeletogenic structures (around the base of the podia). Scale bar: 100 μm. P – proximal, D – distal. Arrowheads – site of developing spines, arrows – site of developing podia, asterisks – intervertebral muscles.
	Fig. 4. Quantification of gene expression during brittle star arm regeneration. (a) The differentiation marker Afi-trop1 shows an upregulation in proximal, differentiated and non-regenerating tissue of the arm. (b,d,e) Afi-ets1/2, Afi-foxB and Afi-alx1 are also highly expressed in distal segments of the 50% and 95% differentiated arms. (c) Afi-gataC is downregulated after the blastema stage. (f) Afi-tbr is not expressed in the adult brittle star; a control using embryonic cDNA stages shows that the primers worked. All other transcription factors are most abundantly expressed in the blastema. Error bars represent standard deviation between two biological replicas (n = 15 animals per cDNA batch). At least four experimental replicas were used for each combination of cDNA and primers in each batch. h - hours post fertilisation.

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