Int J Dev Biol
January 1, 2010;
Comparison of Lim1 expression in embryos of frogs with different modes of reproduction.
A polyclonal antibody was used to detect the expression of the homeodomain protein Lim1
) in embryos of Xenopus laevis, Engystomops randi, Colostethus machalilla and Gastrotheca riobambae. These frogs belong to four separate families, and have differences in their modes of reproduction and developmental rates. The expression of Lim1
in embryos of these frogs resembled the X. laevis expression pattern. Thus, the dorsal blastopore lip
, axial mesoderm
and certain cells of the central nervous system
-positive in embryos of all frogs. There were, however, time differences; thus, in the mid-gastrula
of the rapidly developing embryos of X. laevis and E. randi, the Lim1
protein was simultaneously detected in the prechordal plate
) and notochord
). In contrast, only the prechordal plate
-positive during gastrulation in the slow developing embryos of C. machalilla. The notochord
elongated and became Lim1
-positive after closure of the blastopore
in C. machalilla and G. riobambae embryos. The prechordal plate
of G. riobambae embryos could not be clearly detected, as the Lim1
-signal remained around the blastopore
during gastrulation. These observations indicate that the timing of gene expression at the dorsal blastopore lip
in embryos of slow developing frogs differs from that of X. laevis. Moreover, the comparison shows that the developmental processes of the head
organizers are basically separable and become dissociated in embryos of the slow developing frog, C. machalilla.
Int J Dev Biol
[+] show captions
Fig. 1. Specificity of anti-Xlim1. (A)
Immunoblot analysis. Lanes 1 through 3 are in vitro translated products, and lanes 4 and 5 are embryonic lysates. Lane 2, Xlim1DNK is the N-terminal LIM domain-deleted construct, Xlim1δNK (Karavanov et al., 1996). (B) Whole mount preparation of the G. riobambae noto- chord. The rostral region is oriented towards the top. (C) Higher magnification of the Lim1 signal observed in B. (C Nuclear staining of the image shown in (C). There is quenching of fluorescence in the Lim1-positive nuclei. Three Lim1-positive nuclei are indicated by arrows and a Lim1-negative nucleus by arrowheads in (C,C. In this and the following figures, letters and numbers in the upper right hand corner indicate species and developmental stage. Cm, C. machalilla; Er, E. randi; Gr, G. riobambae; and Xl, X. laevis. Abbreviations: n, notochord. Scale bars: 200 μm (B); 25 μm (C- C.
Fig. 2. Lim1 in the dorsal blas- topore lip. In situ hybridization against Xlim1 mRNA is shown in (A); the remaining embryos were immunostained against Lim1 protein. (A,B) Early gastrulae of X. laevis. (A) Xlim1 mRNA expres- sion. (B) The dorsal blastopore lip in sagittal view reveals the internal Lim1 protein signal. (C,D) Gastrulae of E. randi. (C) Surface Lim1 protein signal in the dorsal blastopore lip. (D) The dorsal blas- topore lip in sagittal view. The dark line in the middle of the image is due to unspecific stain- ing of the blastocoel floor. (E,F) Early gastrulae of C. machalilla. (E) The surface Lim1-signal is visible in the dorsal blastopore lip, and in a belt around the blastopore. (F) The dorsal blastopore lip in sagittal view reveals the internal signal. (G,H) Mid gastrulae of G. riobambae. (G) Lim1 signal at st 12 occurs in a belt around the blastopore with a strong dorsal expression. (H) The blastopore of a st 12 embryo in sagittal view. A higher density of Lim1-positive nuclei were detected in the dorsal blastopore lip in comparison with the ventral lip. Abbreviations: dl, dorsal lip; vl, ventral lip; yp, yolk plug. Scale bars: 200 μm (A,E,G,H); 100 μm (B,C,D,F).
Fig. 3. The notochord and prechordal plate in rapidly developing embryos. (A,A Mid-gastrulae of X. laevis. (A) Whole mount prepara- tion. (A The notochord and prechordal plate in sagittal view. (B,B Mid- gastrulae of E. randi. (B) Whole mount preparation. (B The notochord and prechordal plate in sagittal view. The drawings outline, in black, the Lim1-signal. Abbreviations: a, archenteron; dl, dorsal lip; n, notochord; pp, prechordal plate; yp, yolk plug. Scale bars: 200 μm (A); 100 μm (AB,B.
Fig. 4 (Above). The prechordal plate in slow developing embryos. (A,B Gastrulae of C. machalilla. (A) Mid-gastrula in whole mount. (A Sagittal section of a mid-gastrula. (B) Late-gastrula in whole mount. (B Sagittal section of a late- gastrula. The prechordal plate is Lim1 positive in A-B (C,D) Late gastrulae of G. riobambae. (C) Late-gastrula in whole mount. An image of this embryo at higher magnification was published (del Pino et al., 2007). (D) Sagittal section of a late- gastrula. The dorsal Lim1-positive region may represent the prechordal plate. The drawings outline in black the Lim1 signal; Abbreviations: a, archenteron; b, blasto- pore; cbc, circumblastoporal collar; d, dorsal; dl, dorsal lip; pp, prechordal plate; yp, yolk plug. Scale bars: 500 μm (C); 300 μm in (A); 200 μm in (AB, BD).
Fig. 5. The notochord and pre- chordal plate in the late gas- trula. (A,A X. laevis. (A) Late gastrula in whole mount. (A Sagittal section through the notochord and prechordal plate. (B,B E. randi. (B) Late gastrula in whole mount. (B Sagittal section through the notochord and prechordal plate. (C,C C. machalilla. (C) Late gastrula in whole mount. (C Sagittal sec- tion through the notochord and prechordal plate. (D-D) G. riobambae. (D) Embryo in whole mount. (D Higher magnifica- tion of the notochord from the embryo shown in D. (D) Sagit- tal section through the noto- chord from the embryo shown in D. The drawings outline the
Lim1 signal in black. The down regulation of the protein Lim1 in the prechordal plate of C. machalilla is shown in gray. Abbreviations: a, archenteron; n, notochord; p, pronephros; pp, prechordal plate. Scale bars: 400 μm (C); 300 μm (D,D); 200 μm (ABC; 100 μm (A,B,D.
Fig. 6. Lim1 in the pronephros and cns. (A,A X. laevis neurula. (A) The image is focused in the pronephros. (A Lim1-positive cells of the cns from the embryo in (A). (B,B E. randi neurula. (B) Image focused in the pronephros. (B Lim1-positive cells of the cns from the embryo in (B). (C,C C. machalilla neurula. (C) Image fo- cused in the pronephros. (C Lim1- positive cells of the cns from the embryo in (C). (D,E) G. riobambae neurulae. (D) Lim1 staining of the pronephros and notochord. Cells of the cns were not detected at this stage in G. riobambae em-
bryos. (E) Pronephros and cns Lim1-positive cells from a more advanced embryo. Arrows in ABCand E indicate the Lim1-positive cells of the cns. Abbreviations: n, notochord; p, pronephros, pd, pronephric duct. Scale bars: 400 μm (D); 200 μm (E); 100 μm (A,B,C,C and 50 μm (AB.