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First published online 2 February 2005
doi: 10.1242/dev.01659

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The Notch-target gene hairy2a impedes the involution of notochordal cells by promoting floor plate fates in Xenopus embryos

Silvia L. López^*, María V. Rosato-Siri^*, Paula G. Franco, Alejandra R. Paganelli and Andrés E. Carrasco

Laboratorio de Embriología Molecular, Instituto de Biología Celular y Neurociencias, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155, 3° piso (1121), Buenos Aires, Argentina

View larger version (83K): [in a new window]   Fig. 1. Comparative expression patterns of delta1 and hairy2a. In situ hybridization of (A-B'') delta1, (C-K) hairy2a, (L,M) delta1 (purple) and hairy2a (magenta) in control embryos. Stages (st) are indicated at the bottom of each figure; fp, prospective floor plate; n, notochord; hm, head mesoderm; vmn, delta1 stripe corresponding to the domain of developing primary ventral motor neurons; dotted line, dorsal blastoporal groove. The insets in G and H show low magnifications of the corresponding sagittal sections in G'and H'. See text for details.

View larger version (95K): [in a new window]   Fig. 2. Comparative expression patterns of hairy2a, chd, foxa4a and shh. Double in situ hybridization of (A-A'), chd (turquoise) and hairy2a (magenta); (B,B') foxa4a (magenta) and hairy2a (purple); (D-E'') chd (purple) and foxa4a (magenta); (F-G') shh (turquoise) and hairy2a (magenta). (C,C') in situ hybridization of foxa4a. (A',D',E'',F') Sagittal sections of the embryos shown in A,D,E,F respectively. (G') Transverse section of the embryo shown in G. Notice that in F',G' the co-expression domain of shh and hairy2a in the FP can be distinguished because of the purplish colour resulting from the overlapping of magenta and turquoise staining. Stages (st) are indicated at the bottom of each figure; fp, prospective floor plate; n, notochord; dnimz, dorsal non-involuting marginal zone; dimz, dorsal involuting marginal zone; dotted line indicates the presumptive limit of involution. See text for details.

View larger version (78K): [in a new window]   Fig. 3. Notch signalling up-regulates hairy2a in the DML precursors. delta1 represses the notochordal marker chd and activates the FP marker hairy2a. (A,C) In situ hybridization of hairy2a (purple) in embryos injected with 1 ng of notchICD mRNA fixed at (A) gastrula stage, brown c-myc staining, or (C) at neural plate stage, turquoise c-myc staining. (A',C') Higher magnifications of A and C, respectively. (C'') Transverse section of the embryo shown in C. (B,D) In situ hybridization of hairy2a (purple) in embryos injected with 2 ng of su(H)DBM mRNA fixed at (B) gastrula (turquoise c-myc staining) or (D) at neural plate stages (c-myc brown staining). (B',D') Higher magnifications of B and D, respectively. (D'') Transverse section of another neurula injected with 2 ng of su(H)DBM (magenta c-myc staining), in situ hybridization of hairy2a (purple). (E,F) In situ hybridization of chd (purple) in gastrula stage embryos injected with (E) 1 ng of delta1 mRNA (turquoise X-gal staining) or (F) with 0.5 ng of delta1STU mRNA (turquoise X-gal staining). (E',F') Higher magnifications of E and F, respectively. (G,H) In situ hybridization of hairy2a (purple) in gastrula stage embryos injected with (G) 1 ng of delta1 mRNA (turquoise X-gal staining) or (H) with 0.5 ng of delta1STU mRNA (turquoise X-gal staining). (G',H') Higher magnifications of G and H, respectively. Green asterisks indicate increase effects and red asterisks, decreased effects. In all embryos the injected side is oriented to the right.

View larger version (72K): [in a new window]   Fig. 4. hairy2a down-regulates the notochordal markers chd and bra and impairs involution of notochordal cells. (A-D) chd expression (purple) in embryos injected with 1 ng of hairy2a mRNA (turquoise X-gal staining). (A) Dorsally injected gastrula. (B) Ventrally injected gastrula. The yellow arrowhead points to the ectopic chd+ cells. (C) Dorsally injected embryo, fixed when sibling controls reached the neurula stage. (D) Ventrally injected neurula. The white arrow points to the normal axis expressing chd. The yellow arrowhead points to the ectopic chd+ cells, which were unable to involute. (E) chd expression (purple) in a gastrula injected with 10 ng of AMOh (turquoise X-gal staining). (E') Higher magnification of E. (E'',E''') chd expression (purple) in parasagittal sections of another gastrula injected with 10 ng of AMOh (whole embryo shown in the inset in E'''; yellow lines e'',e''' indicate the levels of the sections shown in E'',E''', respectively). (E'') Non-injected side. (E''') Injected side. The turquoise dots reveal the lacZ tracer. (F) chd expression (purple) in a late gastrula injected with 0.5 ng of hairy2a mRNA plus 10 ng of AMOh. The magenta staining reveals the distribution of the injected AMOh. (F') Higher magnification of F. (G) bra expression in a control gastrula, vegetal view. (G') Ventrally tilted view of the same embryo shown in G. (H,I) bra expression (purple) in gastrula stages embryos (H) dorsally or (I) ventrally injected with 0.25 ng of hairy2a mRNA (turquoise X-gal staining). (H') Higher magnification of H. (J,K) bra expression (purple) in embryos injected with 10 ng of AMOh fixed at (J) early gastrula, magenta AMO staining, or (K) late gastrula, turquoise AMO staining. (J',K') Higher magnifications of J and K, respectively. Green asterisks indicate increase effects and red asterisks, decrease effects. In all embryos the injected side is oriented to the right.

View larger version (82K): [in a new window]   Fig. 5. hairy2a up-regulates foxa4a and shh in the FP precursors. (A) foxa4a expression (purple) in a gastrula dorsally injected with 0.25 ng of hairy2a mRNA (turquoise X-gal staining). (A') Higher magnification of A, revealing the increase of foxa4a+ cells in the notoplate precursors (green asterisk). (A'') foxa4a expression (purple) in a gastrula ventrally injected with 1 ng of hairy2a mRNA (turquoise X-gal staining). The arrowhead points to the ectopic foxa4a+ cells in the ventral mesoderm region. (B,C) foxa4a expression (purple) in (B) gastrula or (C) neural plate stage embryos injected with 10 ng of AMOh, turquoise staining reveals the distribution of AMOh. (B',C') Higher magnification of B and C, respectively. In B' the yellow lines indicate the plane of the parasagittal sections shown in B'' and B''', respectively. (C'') Transverse section of the embryo shown in C. (D) shh expression (purple) in a neurula injected with 0.25 ng of hairy2a mRNA (turquoise X-gal staining). (D') Higher magnification of D, revealing the increase of shh+ superficial cells corresponding to the prospective FP. (D'') Transverse section of the same embryo shown in D. Notice the increase of shh+ cells in the FP domain on the injected side (green asterisk). (E) shh expression (purple) in a neurula injected with 10 ng of AMOh, turquoise staining reveals the distribution of AMOh. (E') Higher magnification of E, revealing the decrease of shh+ superficial cells corresponding to the prospective FP. (E'') Transverse section of the embryo shown in E. Notice the decrease of shh+ cells in the FP domain on the injected side (red asterisk). (F) Expression of chd (purple) and foxa4a (magenta) in a late gastrula embryo injected with 0.25 ng of hairy2a mRNA (turquoise X-gal staining). nis, non-injected side; is, injected side; fp, prospective floor plate; n, notochord. The dotted red line demarcates the notochord. See text for details.

View larger version (72K): [in a new window]   Fig. 6. hairy2a mediates the cell-fate switch executed by Notch in the DML precursors. (A-C) chd expression (purple) in gastrulae injected with (A) 1 ng of notchICD mRNA (magenta c-myc staining), (B) 10 ng of AMOh (magenta staining) or (C) 1 ng of notchICD mRNA (magenta c-myc staining) plus 10 ng of AMOh. (D-F) foxa4a expression (purple) in gastrulae injected with (D) 1 ng of notchICD mRNA (magenta c-myc staining), (E) 10 ng of AMOh (turquoise staining) or (F) 1 ng of notchICD mRNA (turquoise, c-myc staining) plus 10 ng of AMOh. (D'-F') foxa4a expression (purple) in late gastrula/early neurula embryos injected with (D') 1 ng of notchICD mRNA (magenta c-myc staining), (E') 10 ng of AMOh (turquoise staining) or (F') 1 ng of notchICD mRNA (turquoise c-myc staining) plus 10 ng of AMOh. (G-I) shh expression (purple) in neurula embryos injected with (G) 1 ng of notchICD mRNA (magenta c-myc staining), (H) 10 ng of AMOh (turquoise staining) or (I) 1 ng of notchICD mRNA (magenta c-myc staining) plus 10 ng of AMOh. (G'-I') Transverse sections of the embryos shown in G-I. fp, prospective floor plate; n, notochord. The dotted red line demarcates the notochord. Green asterisks indicate increase effects and red asterisks, decrease effects. In all embryos the injected side is oriented to the right.

View larger version (50K): [in a new window]   Fig. 7. Model interpreting the Notch-mediated cell-fate switch in the DML precursors. (Left) hairy2a expression (purple) in a developmental series of gastrula embryos injected with 1 ng of notchICD mRNA (brown or turquoise c-myc staining): left column, whole embryos; right column, higher magnification of the notoplate region. In all embryos the injected side is oriented to the right. (Right) Scheme illustrating the molecular and cellular changes on DML precursors after notchICD mRNA injection. See text for details. e, prospective epidermis; na, neural anlage; np, notoplate; n, notochord; ph, pharyngeal mesendoderm; white arrows, convergent-extension movements.