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First published online August 4, 2003
doi: 10.1242/10.1242/dev.00629

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Feedback loops comprising Dll1, Dll3 and Mesp2, and differential involvement of Psen1 are essential for rostrocaudal patterning of somites

Yu Takahashi1, Tohru Inoue1, Achim Gossler2 and Yumiko Saga3,*

1 Cellular and Molecular Toxicology Division, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagayaku, Tokyo 158-8501, Japan
2 Institut für Molekularbiologie, MHH, 30625 Hannover, Germany
3 Division of Mammalian Development, National Institute of Genetics, SOKENDAI, Yata 1111, Mishima 411-8540, Japan



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  		Fig. 1. Positive and negative feedback loops of Dll1 and Mesp2 are essential for stripe formation. (A-F) Dll1 induces expression of Dll1 itself. Expression of Dll1-lacZ mRNA was detected by in situ hybridization in Dll1+/L (A-C) and Dll1L/L (D-F) embryos at 9.5 dpc. (A,D) Lateral view, (B,E) dorsal view of the tail region. (C,F) Transverse section at the anteriormost PSM. In the Dll1+/L embryo, lacZ expression reflects normal stripe pattern of Dll1, localized at the caudal half of somites (arrowheads in B). In the Dll1L/L embryo, the stripe of Dll1-lacZ is lost at the putative somite region (anterior to the arrow in D). Ectopic strong staining in the ventral neural tube is evident (F). (G-J) Expression of Mesp2 is severely decreased in the Dll1-null embryo (G,H) while expression of Dll1 is strongly expanded in the Mesp2-null embryo (I,J). (K-Q) Mesp2-lacZ mRNA (with Dll1-lacZ in case of the double mutant) was detected by in situ hybridization. (K-M) Dorsal views and (N-Q) lateral views. After extended staining, Dll1-lacZ expression appears at the neural tube and the PSM, but not at the somite region (Q, arrow indicates the putative boundary between PSM and somite region). (R) Summary of reciprocal regulation of Dll1 and Mesp2. In the absence of Dll1, both Dll1 stripes and normal level of Mesp2 expression are lost. In the absence of Mesp2, both Dll1 and Mesp2-lacZ expressions are strongly expanded. The Dll1/Mesp2 double-null embryo is similar to the Dll1-null embryo in terms of reciprocal regulation.

 


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  		Fig. 5. Dll1-Notch signaling consists of both Psen1-dependent and Psen1-independent pathways. Normal Uncx4.1 expression (A) is lost in both Dll1 (B) and Psen1-null (C) embryos, as well as in Dll1/Psen1 double-null embryo (D). The stripe expression of the rostral marker Cer1 (E) is almost lost in the Dll1-null embryo (F), whereas it is expanded in the Psen1-null embryo (G). This expanded expression is lost by the additional loss of Dll1 (the Dll1/Psen1 double-null embryo, H). Likewise, Mesp2 expression (I) is almost lost in the Dll1-null embryo (J), moderately reduced in the Psen1-null embryo (K) and is almost lost in the Dll1/Psen1-double null embryo (L). (M-P) Mesp2 expression is partly dependent on Psen1-dependent Dll3-Notch signaling. When compared with the wild type (M), expression levels of Mesp2 are decreased in the Dll3-null (N), Psen1-null (O) and Dll3/Psen1 double-null (P) embryos, and they are comparable among the three genotypes.

 


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  		Fig. 2. Dll1 is required for normal expression of both rostral and caudal genes, and Mesp2 suppresses the caudal half property in both Dll1-dependent and Dll1-independent manners. Expression of Cer1 is usually observed as two or three stripes, finally localizing to the rostral half of nascent somite in the wild-type embryo (A). Cer1 expression is almost lost in both Dll1-null and Mesp2-null embryos (B,C), as well as the Dll1/Mesp2 double-null embryo (D). Normal stripes of Uncx4.1 expression, localizing to the caudal half of each somite (E), are completely lost in the Dll1-null embryo (F). In Mesp2-null embryos, expression of both Dll1 (Fig. 1) and Uncx4.1 is strongly expanded (G). However, the additional loss of Mesp2 in the Dll1-null embryo results in an expanded pattern of Uncx4.1 expression (H). Genetic cascades are also shown.

 


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  		Fig. 3. Dll3 and Mesp2 are required for normal expression of each other. In the wild-type embryo at 9.5 dpc, expression of Dll3 is finally localized to the rostral half of each somite (A). The Dll3 stripe (arrowhead in A) is missing in the Dll3pu/pu embryo (B). The level of Mesp2 expression is significantly decreased in the Dll3pu/pu embryo (C,D). In the Mesp2-null embryo, a weak diffuse Dll3 expression is expanded rostrally (E,F). (G-J) Expansion of Dll3 expression in the Mesp2-null embryo does not require Dll3. At 11.5 dpc, in the Dll3pu/pu embryo, the Dll3 stripe is missing and the expression is not expanded rostrally (G,H). Expansion of Dll3 expression in the Mesp2-null embryo is not largely affected by the loss of Dll3 (I,J). (K,L) Dll3 is required for localization of Mesp2 expression into the rostral half of somites. In the wild type, ß-gal activity for Mesp2-lacZ is localized in the rostral half of somites (K). A randomized salt-and-pepper pattern is observed in the Dll3pu/pu embryo (L).

 


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  		Fig. 4. Genetically, Mesp2 lies downstream of Dll3 regarding the rostrocaudal polarity. Expression of the caudal genes Dll1 (A-D), Uncx4.1 (E-H) and the morphology of the lumbar vertebrae (I-L) are examined in the Dll3/Mesp2 intercross. Genotypes are indicated on the left. The Dll3/Mesp2 double-null embryo exhibits phenotypes indistinguishable from those of the Mesp2-null embryo. Details are stated in the text. For the rostral genes, see Fig. S1 at http://dev.biologists.org/supplemental/.

 


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  		Fig. 6. Dll3 and Psen1 can act independently of each other in regulation of the caudal genes. The stripe pattern of Dll1 and Uncx4.1 is correlated with the skeletal morphology of the vertebrae (A,F,K). In the Dll3pu/pu embryo, the blurred and randomized expression of Dll1 and Uncx4.1 results in disorganized skeletal elements (B,G,L). In the Psen1-null embryo (Dll3+/+Psen1-/-), stripes of Dll1 and Uncx4.1 expression, and the pedicles were completely lost (C,H,M). Weak disorganized expression of Uncx4.1 was observed in the Dll3/Psen1 double-null embryo (Dll3pu/puPsen1-/-; D,I). The vertebrae of Dll3pu/puPsen1-/- exhibited an intermediate morphology between Dll3-null and Psen1-null vertebrae (N). Surprisingly, the Dll3+/puPsen1-/- embryo exhibited faint stripes of Dll1 (E, arrowheads) and Uncx4.1 (J), and a small amount of skeletal elements (O, arrowheads).

 


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  		Fig. 7. (A) Summary of putative signaling cascades in the anterior PSM. The Psen1-independent pathways are shown with green arrows. Dll1-Notch signaling results in induction of both Dll1 itself and Mesp2. The positive feedback of Dll1 is mediated by the Psen1-dependent signal. Induction of Mesp2 is mediated via Psen1-independent Dll1-Notch signaling and Psen1-dependent Dll3-Notch signaling. In contrast to Dll1, Dll3 has roles in upregulation of Mesp2 and suppression of Dll1 and Uncx4.1. (B) Integration of stripe pattern by Dll3 function. For simplification, anterior PSM cells of four-cell width are illustrated. Pink cells represent the dominance of the Mesp2 function, and blue cells the dominance of the Dll1 function. Genes and arrows are shown only between two representative cells for simplicity. The green arrows show the Psen1-independent pathways and broken lines show inactive states. Even in the absence of Dll3, Dll1-Notch signaling and Mesp2 are still active (left). Reciprocal Dll1-Notch signaling between two neighboring cells results in induction of Dll1 in both cells. Meanwhile, reciprocal Dll1-Notch signaling also induces expression of Mesp2, which suppresses expression of Dll1 cell-autonomously in both cells. When Dll1 is downregulated, Mesp2 is also reduced by the lack of the juxtacrine Dll1 signal. Thus, the positive and negative feedback loops of Dll1 and Mesp2 produce uneven spatial patterns of Dll1 and Mesp2, but fail to form integrated stripe patterns in the absence of Dll3. Although the precise mechanism is unknown, participation of Dll3 results in synchronization of Dll1-dominant and Mesp2-dominant cells by suppressing Dll1 expression in cooperation with Mesp2 (right). After segregation, Dll3 and Mesp2 continue to suppress Dll1 and Uncx4.1 expression in the rostral half, while Dll1 induces expression of Dll1 itself and Uncx4.1 via Psen1-dependent pathway in the caudal half. In the caudal half, induction of Mesp2 expression via Psen1-independent pathway is inactive.

 

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© The Company of Biologists Ltd 2003