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Comparative Study
. 2010 Jun;216(6):683-91.
doi: 10.1111/j.1469-7580.2010.01233.x. Epub 2010 Apr 14.

Comparison of Iroquois Gene Expression in Limbs/Fins of Vertebrate Embryos

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Free PMC article
Comparative Study

Comparison of Iroquois Gene Expression in Limbs/Fins of Vertebrate Embryos

Laura A McDonald et al. J Anat. .
Free PMC article

Abstract

In Drosophila, Iroquois (Irx) genes have various functions including the specification of the identity of wing veins. Vertebrate Iroquois (Irx) genes have been reported to be expressed in the developing digits of mouse limbs. Here we carry out a phylogenetic analysis of vertebrate Irx genes and compare expression in developing limbs of mouse, chick and human embryos and in zebrafish pectoral fin buds. We confirm that the six Irx gene families in vertebrates are well defined and that Clusters A and B are duplicates; in contrast, Irx1 and 3, Irx2 and 5, and Irx4 and 6 are paralogs. All Irx genes in mouse and chick are expressed in developing limbs. Detailed comparison of the expression patterns in mouse and chick shows that expression patterns of genes in the same cluster are generally similar but paralogous genes have different expression patterns. Mouse and chick Irx1 are expressed in digit condensations, whereas mouse and chick Irx6 are expressed interdigitally. The timing of Irx1 expression in individual digits in mouse and chick is different. Irx1 is also expressed in digit condensations in developing human limbs, thus showing conservation of expression of this gene in higher vertebrates. In zebrafish, Irx genes of all but six of the families are expressed in early stage pectoral fin buds but not at later stages, suggesting that these genes are not involved in patterning distal structures in zebrafish fins.

Figures

Fig. 1
Fig. 1
An unrooted Bayesian phylogram of Iroquois genes. Numbers indicate support values. Note that each of the six Irx genes form well defined groupings, e.g. Irx1 gene family is well defined with all genes in expected phylogenetic locations. Chick Irx5 is misidentified, as it belongs to Irx6 groupings. Species are as follows: Hs, Homo sapiens; Mm, Mus musculus; Bt, Bos taurus; Cf, Canis familiaris; Gg, Gallus gallus; Dr, Danio rerio; Xl, Xenopus laevis; Rn, Rattus norvegicus.
Fig. 2
Fig. 2
mIrx1-6 expression in E11.5–E14.5 mouse limbs. mIrx1 and mIrx2 have the same pattern of expression in limbs throughout development, although mIrx2 expression is much weaker. (A,E) mIrx1 and mIrx2 are proximally expressed in forelimb and hindlimb at E11.5. (B,F) Expression of mIrx1 and mIrx2 is restricted to condensing digits at E12.5, with stronger expression in digits 2–4. (C,G,D,H) mIrx1 and mIrx2 are expressed in joint-forming regions at E13.5 and E14.5. (I,J) mIrx3 is expressed at the distal edge of both forelimb and hindlimb at E11.5 and E12.5. (K,L) Strong expression of mIrx3 at distal edges of digits at E13.5 and E14.5 and also interdigitally at E13.5. (M) mIrx4 distally expressed at E11.5. (N) At E12.5, mIrx4 expression is reduced; expression does not reach the tip and appears to be weakly expressed in proximal interdigital regions. (O) mIrx4 strong in interdigital regions at E13.5, and then at the distal edges of the digits at E14.5 (P). (Q) mIrx5 stronger in distal limb regions at E11.5. (R) At E12.5, mIrx5 expression very weak, but faint expression can be seen in forelimb interdigits. (S) mIrx5 restricted to interdigital regions at E13.5, with stronger expression around digit tips in the forelimb. (T) At E14.5, mIrx5 expressed between and around the edges of the digits. (U) mIrx6 expressed in the middle of forelimb and proximally in hindlimb at E11.5. (V) Weak mIrx6 expression seen at E12.5 in region just proximal to digital plate. (W,X) mIrx6 expression in proximal interdigital regions at E13.5 and E14.5.
Fig. 3
Fig. 3
cIrx1-6 expression in chick limbs. (A–C) cIrx1 expressed proximally in HH24–26 limbs, with distal expression initiated posteriorly in hindlimbs at HH25. (D) cIrx1 is initiated in distal wing at HH27 posteriorly, whilst Irx1 in leg has extended anteriorly at this stage to become expressed in two spots. (E,F) cIrx1 expression extends anteriorly in wings and legs, becoming more strongly expressed in developing digits at HH29. (G,H) At late stages of digit development (HH30/31), expression of cIrx1 is restricted to anterior digits. (I,J) cIrx2 is expressed distally in both wing and leg at HH24/25, with high expression developing at anterior and posterior edges of the limbs between HH26 and 28 (K–M). (N) cIrx2 is strongly expressed in distal digit-forming regions at HH29. (O) cIrx3 is highly expressed in distal limb at early stages before becoming restricted to the outside edges of the limbs at HH25 (P). (Q–T) From HH26 to 29, cIrx3 expression is confined to the distal rim of wing and leg. (U–W) cIrx4 expression is not detected in limbs from HH24 to 26. (X,Y) Expression of cIrx4 is detected in the distal limb at HH27 and HH28. (Z) At HH29, strong expression of cIrx4 is seen distally in both wing and leg. (a–c) Like cIrx4, cIrx6 expression is not detected in early (HH24–26) limb buds. (d) At HH27, anterior and posterior regions of expression in wing and in posterior interdigital regions in leg. (e) cIrx6 strongly expressed in leg interdigital regions, with weaker expression in wing interdigital regions at HH28.
Fig. 4
Fig. 4
Section in situ hybridization of Irx1 in chick, mouse and human limbs. (A) At HH27, cIrx1 is expressed in the posterior digit condensation(s) in wing and leg. (B) At HH28, cIrx1 is not expressed throughout the cartilage digit rudiments but is expressed at the edges in both wing and leg and in developing joints. (C) mIrx1 strongly expressed in all digit condensations of forelimb and hindlimb at E12.5. (D) At E13.5, expression in hindlimb throughout the skeleton of digits 1 and 5 and in joint-forming regions of digits 2, 3 and 4; in the more developmentally advanced forelimb, expression in joint-forming regions. Absence of digit 5 in hindlimb at E12.5 and digit 5 in forelimb at E13.5 is due to plane of section; additional serial sections showed Irx1 expression in these digits. (E) hIrx1 expressed in digits 2–5 of the hindlimb at CS18. (F) At CS19, expression in joint-forming regions of digits (arrows). Absence of digit 1 in E and F due to plane of section; additional serial sections showed expression in digit 1.
Fig. 5
Fig. 5
Zebrafish Iroquois pectoral fin bud expression at 30, 36 and 48 hpf. Position of pectoral fin buds indicated by white-dashed circles; arrows indicate expression in pectoral fins. At 36 hpf, expression of six of 11 zIrx genes detected in pectoral fins. (A’) zIrx1a faintly expressed in pectoral fins at 36 hpf. (C’,D’,F’, respectively) zIrx2a, zIrx3a and zIrx4a expressed throughout developing pectoral fins (white arrows). (H’) zIrx5a displays strongest pectoral fin expression (arrows in H’). (I’) zIrx5b also expressed in pectoral fins, albeit with a weaker expression than zIrx5a. (H’’) By 48 hpf, only zIrx5a shows pectoral fin expression (weak expression, arrow in H’’). The 10 remaining zIrx genes are not expressed in pectoral fins at 48 hpf (A’’–G’’,I’’–K’’).

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