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First published online 15 October 2003
doi: 10.1242/dev.00811

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The amphioxus Hairy family: differential fate after duplication

Carolina Minguillón^1,*, Senda Jiménez-Delgado¹, Georgia Panopoulou² and Jordi Garcia-Fernàndez^1,

¹ Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, Avenida Diagonal 645, E-08028 Barcelona, Spain
² Evolution and Development Group, Depart. Prof. Hans Lehrach, Max-Planck-Institut fur Molekulare Genetik, 14195 Berlin (Dahlem), Germany

View larger version (45K): [in a new window]   Fig. 1. The amphioxus Hairy subfamily. (A) Alignment of the amphioxus Hairy proteins conducted by ClustalW. Black boxes represent identical residues among the Amphi-Hairy proteins. The bHLH, the orange and the C-terminal WRPW domains are indicated. (B) Neighbour-joining phylogenetic tree relating the amphioxus Hairy proteins with other selected Hairy proteins from vertebrate and protostome species. The tree is rooted using the human and the mouse Hes2 sequences. The numbers refer to the bootstrap values. c, chicken; Dm, Drosophila melanogaster; h, human; m, mouse; Tc, Tribolium castaneum; X, Xenopus laevis; zf, zebrafish.

View larger version (47K): [in a new window]   Fig. 2. Linkage of Amphi-hairyA to Amphi-hairyF genes in the amphioxus genome. DNA from PAC 51K5 was subjected to PCR reactions with Amphi-Hairy specific oligonucleotides. Amphi-Hairy A to Amphi-Hairy F (lines A to F) gave positive bands of the expected sizes (Amphi-hairyA, 300 bp; Amphi-hairyB, 156 bp; Amphi-hairyC, 311 bp; Amphi-hairyD, 249 bp; Amphi-hairyE, 235 bp; Amphi-hairyF, 207 bp), indicating that they are contained in the same genomic region.

View larger version (71K): [in a new window]   Fig. 3. Amphi-hairyA, Amphi-hairyB, Amphi-hairyC and D genes expression in amphioxus gastrulae by whole-mount in situ hybridisation. All embryos in dorsal views (A-C,E-G) have anterior towards the left. The lateral view (H) has anterior towards the left and dorsal upwards. In the transverse optical dissection (D), dorsal is upwards looking from a posterior position. The presumptive somitic bands are numbered and the arrows indicate expression domains in the neural plate (with the exception of D). (A) Amphi-hairyA expression within the anterior inner endodermal cell layer and the posterior neural plate. (B) Dorsal view of an Amphi-hairyB two- to three-stripe gastrula. (C) Dorsal view of an Amphi-hairyB three-stripe embryo. (D) Optical transverse dissection of the specimen in C showing the highest Amphi-hairyB expression levels at the edges of the neural plate (asterisks). The expression in the dorsolateral walls of the archenteron is shown by arrows. (E) Dorsal view of an Amphi-hairyB 4-stripe pattern embryo. (F) Dorsal view of a three-stripe pattern embryo stained for the Amphi-hairyC probe. (G) Dorsal view of a four-stripe embryo stained for the Amphi-hairyD labelled probe. (H) Lateral view of the gastrula in G showing the Amphi-hairyD gene expression as pre-somitic stripes.

View larger version (137K): [in a new window]   Fig. 4. Amphi-hairyA, Amphi-hairyB, Amphi-hairyC and Amphi-hairyD expression in amphioxus neurulae by whole-mount in situ hybridisation. Lateral views (A-D,H,J,N,R,V) have anterior towards the left and dorsal upwards. In dorsal views (I and O), anterior is towards the left. In transverse sections (E-G,K-M,P,Q,S,T,W,X), dorsal is upwards looking from a posterior direction. (A) Amphi-hairyA expression in the endoderm and the posterior dorsal nerve cord (arrow) of an early amphioxus neurula. (B) Amphi-hairyA expression in the endoderm and the posterior dorsal nerve cord (arrow) of a late neurula. The expression in the Hatschek's left diverticulum is shown by arrowheads in A and B. (C) Oblique view of an early neurula that exhibits Amphi-hairyB expression in the whole neural plate and the posterior compartment of the segmented somites (the last somitic border is marked by an arrow). (D) Lateral view of an early neurula showing Amphi-hairyB expression in the neural tube, the posterior PSM and the segmented somites. (E) Cross-section through level i in D showing the expression of Amphi-hairyB in the neural tube (asterisk). (F) Cross-section through level ii in D showing the expression of Amphi-hairyB in the neural tube (asterisk) and the segmented somites (arrows). (G) Cross-section through level iii in D showing the expression of Amphi-hairyB in the posterior PSM (arrows). (H) Lateral view of late neurula showing Amphi-hairyB expression in the neural tube and the posterior PSM (arrow). (I) Dorsal view of the specimen in H; the expression in the PSM is shown by arrows. The gap in the anterior neural tube domain is shown by a line in H and I. (J) Lateral view of an early neurula stained for the Amphi-hairyC mRNA with the expression in the neural plate, the somites and the posterior PSM. (K) Cross-section through level i in J showing the expression of Amphi-hairyC in the neural plate (arrowhead) and the anterior gut (arrows). (L) Cross-section through level ii in J showing the expression of Amphi-hairyC in the neural tube (arrowhead) and the somites (arrows). (M) Cross-section through level iii in J showing the expression of Amphi-hairyC in the posterior neural plate (arrowhead) and mesoderm. The low expression levels in the forming notochord is shown by an asterisk. (N) Lateral view of a late neurula stained for the Amphi-hairyC mRNA showing the expression in the neural plate, the anterior gut, the somites and the posterior PSM. (O) Dorsal view of the specimen shown in N. The arrows indicate the expression in the PSM. (P) Cross-section through level i in N showing the expression of Amphi-hairyC in the neural tube (arrowhead) and the segmented somites (arrows). The low expression in the dorsal notochord is shown by an asterisk. (Q) Cross-section through level ii in N showing the expression of Amphi-hairyC in the posterior mesoderm and no longer in the neural plate (asterisk). (R) Lateral view of an early neurula showing Amphi-hairyD expression in the anterior endoderm, the neural tube and the notochord. (S) Cross-section through level i in R showing the expression of Amphi-hairyD in the neural plate (arrowhead) and the gut (arrow and double arrow). (T) Cross-section through level ii in R showing the expression of Amphi-hairyD in the posterior mesoderm (arrow). The lack of Amphi-hairyD expression in the posterior neural plate is shown by an asterisk. (V) Lateral view of a late neurula showing Amphi-hairyD expression in left gut diverticulum (arrow), the neural tube and the notochord. (W) Cross-section through level i in V showing the expression of Amphi-hairyD in the neural plate (arrowhead), the ventral notochord (asterisk) and the gut (arrow). (X) Cross-section through level ii in V showing the expression of Amphi-hairyD in the posterior mesoderm. The asterisk marks the expression in the forming notochord.

View larger version (53K): [in a new window]   Fig. 5. Amphi-hairyA, Amphi-hairyB, Amphi-hairyC and Amphi-hairyD genes expression in amphioxus larvae by whole-mount in situ hybridisation. Lateral views (A,D,F,J) have anterior towards the left and dorsal upwards. In dorsal and ventral views (I,K,N), anterior is towards the right. In transverse sections (B,C,E,G,H,L,M), dorsal is upwards looking from a posterior position. (A) Amphi-hairyA expression in the endoderm and the posterior dorsal nerve cord of an amphioxus larva. Scattered positive cells are in the middle gut are indicated by a double arrow and expression in the left anterior gut diverticulum is indicated by an arrow. (B) Cross-section through level i in A showing the expression of Amphi-hairyA in the preoral pit (arrow). (C) Cross-section through level ii in A showing the expression of Amphi-hairyA in the gut (arrow) and the posterior neural tube (asterisk). (D) Amphi-hairyB expression in the anterior half of the dorsal nerve cord. The gap in the anterior neural tube domain is shown by a double arrow. (E) Cross-section through level i in D showing the expression of Amphi-hairyB in the neural tube (asterisk). (F) Lateral view of an amphioxus larva showing Amphi-hairyC expression in the posterior tail bud, and in the anterior endoderm (arrow indicates the pharyngeal endoderm, and asterisk indicates the preoral pit). (G) Cross-section through level i in F showing the expression of Amphi-hairyC in pharyngeal endoderm (arrow). (H) Cross-section through level ii in F showing the expression of Amphi-hairyC in the posterior mesoderm. (I) Dorsal view of the specimen in F showing the Amphi-hairyC expression within the tail bud. (J) Lateral view of a larva showing Amphi-hairyD expression in the anterior club-shaped gland (asterisk) and the presumptive first and second gill slits (arrows), and in the posterior mesoderm. (K) Ventral view of the specimen in J. (L) Cross-section through level i in J showing the expression of Amphi-hairyD in the club-shaped gland (arrow). (M) Cross-section through level ii in J showing the expression of Amphi-hairyD in the posterior mesoderm. (N) Dorsal view of the specimen in J showing the Amphi-hairyD expression within the tail bud.

View larger version (74K): [in a new window]   Fig. 6. RT-PCR on RNA obtained from amphioxus embryonic stages. 12, 15, 18 and 21 refer to hours after fertilisation at 25°C.–, negative control; C, Amphi-hairyC positive control; E-H, Amphi-hairyE to Amphi-hairyH positive controls. The expected size and the name of the amplified bands are indicated. Amphi-hairyE (A), Amphi-hairyF (B), Amphi-hairyG (C) and Amphi-hairyH (D) are not expressed during those stages or are expressed at undetectable levels. For Amphi-hairyH the positive control gives a larger size than the expected in the RT-PCR as the oligonucleotides for Amphi-hairyH expand over two small introns, and the template for the positive control is a plasmid containing genomic regions.

View larger version (24K): [in a new window]   Fig. 7. Subfunctionalisation of amphioxus Hairy genes after duplication. (A) Summary of Amphi-Hairy gene expression during amphioxus embryogenesis in a dorsal flattened view of an idealised half amphioxus embryo. The four main embryonic tubes are indicated [from dorsal (D) to ventral (V): nerve cord (N), notochord (nt), paraxial mesoderm (pM), gut (g)]. Each Amphi-Hairy gene expression is indicated by color code and line patterns. The summation of Amphi-hairyA to Amphi-hairyD expression mimics that of the single (mouse) or the two (chicken, frog) Hairy genes. (B) Schematic representation of representative enhancers deduced for Amphi-Hairy before gene duplication in the amphioxus lineage. After duplication, specific enhancers were maintained in each copy of Amphi-hairyA to Amphi-hairyF. end1-3, distinct anteroposterior regions of the endoderm; NT1-4, distinct anteroposterior regions of the neural tube; nt, notochord; PSM, presomitic mesoderm.