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First published online 15 September 2004
doi: 10.1242/dev.01389

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Targeted deletion of the novel cytoplasmic dynein mD2LIC disrupts the embryonic organiser, formation of the body axes and specification of ventral cell fates

Amer Ahmed Rana^1,*,**, Juan Pedro Martinez Barbera^1,, Tristan A. Rodriguez^1,, Denise Lynch^1,, Elizabeth Hirst¹, James C. Smith^2,* and Rosa S. P. Beddington^1,¶

¹ Division of Mammalian Development, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
² Division of Developmental Biology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK

View larger version (61K): [in a new window]   Fig. 3. Targeted deletion of mD2LIC. (A) The targeting strategy involved removing 2.75 kb of the endogenous mD2LIC locus. This contains 500 bp upstream of the predicted transcriptional start site together with the first two exons, which include the translation start site and half the P-loop domain. The targeting vector incorporates both positive (neomycin resistance) and negative (diphtheria toxin A) selection, and comprises a 5.5 kb 5' homology arm, a 2 kb loxP flanked PGKneo cassette (which replaces the targeted region) and a 2.3 kb 3' homology arm. During the targeting event, an exogenous HindIII restriction site is introduced into the locus just downstream of the PGKneo cassette. (B) Use of a 3' probe in Southern blot analyses of transfected ES cell genomic DNA digested with HindIII reveals a 13 kb band corresponding to the wild-type locus and a 6 kb band representing a correctly targeted locus. (C) Northern blot analysis of littermates from heterozygous crosses demonstrates loss of the mD2LIC transcript in homozygous mutant individuals and a decrease in heterozygous animals. (D) Primers P1 and P2 (indicated in A), together with a pair of primers designed to detect neo, distinguish between targeted and non-targeted alleles when genotyping. (E) Classification of mD2LIC–/– mutants into three groups according to the extent of embryonic turning at 9.5 dpc (Table 1). Class I mutants (bottom, 61%; n=39), which exhibit the most severe phenotype, fail to initiate embryonic turning. Three examples are shown here. Class II (middle, 21%) mutants start but do not complete embryonic turning, and Class III (top, 18%) mutants complete turning but always display an open neural tube in the region of the head (arrowhead). Other defects include reversal of heart looping (Class II, white arrowhead), ballooning of the pericardial sac, anterior truncations (Class III, white arrowhead) and defects in truck and tail development.

View larger version (89K): [in a new window]   Fig. 1. cDNA sequence and translation of mD2LIC. The cDNA comprises 1390 bp and codes for a 351 amino acid protein predicted to contain a P-loop (nucleotide binding domain) near its N terminus (red box). This sequence differs from the previously published mD2LIC (Grissom et al., 2002), but the predicted protein resembles closely the rat and human gene products. We suspect that the previously published sequence contains a frame-shift near the 5' end of the cDNA, resulting in a predicted protein of only 209 amino acids that lacks the P loop.

View larger version (99K): [in a new window]   Fig. 2. Expression pattern of mD2LIC. Transcription of mD2LIC is not detected by in situ hybridisation before the onset of gastrulation (A), but from late streak (B) to headfold stages (C,D) transcription is observed in the node (black arrowheads). Sections reveal that this expression is restricted to cells of the ventral node (E). Northern blot analysis reveals a single transcript of 1.4 kb, and in the adult expression is highest in brain and kidney (E).

View larger version (166K): [in a new window]   Fig. 4. Scanning electron microscopy reveals defects in monocilium formation in mD2LIC–/– mouse embryos. (A) The distal tip of a wild-type embryo at 7.5-8.0 dpc (1-2 somite stage) viewed at low power. (C,E) The gross morphology of the node of homozygous mutant embryos is normal. Conditions and magnification are identical in A and C, and E is at twice the magnification. (B,G) Higher-power view of a wild-type embryo reveals rounded cells bearing monocilia (arrowheads). (D,H,F,I) Ventral node cells in mD2LIC–/– embryos are flatter than their wild-type counterparts and they lack normal monocilia. In some cases stunted structures are formed in the place of monocilia (arrows). G-I are at the same magnification. White boxes in B,D,F indicate regions of the node shown at higher magnification in G-I respectively. (F) Cells in the anterior region of the node and notochordal plate have the most extreme phenotype.

View larger version (113K): [in a new window]   Fig. 5. Analysis of the mD2LIC phenotype at gastrula stages. (A,B) Expression of Fgf8 is unaffected by loss of mD2LIC function. (A) Lateral view of wild-type embryo; (B) Lateral view of mD2LIC–/– embryo. (C-E) Shh expression is reduced in the node and axial mesoderm of mD2LIC-null mutants. (C) Wild-type embryo showing expression of Shh in the node and in axial structures. (D,E) Reduction of Shh expression (arrowheads) in mD2LIC–/– embryos. (F-J) Reduction in Foxa2 expression in mD2LIC null mutant embryos. (F) Wild-type embryo showing expression of Foxa2 in the node and axial mesendoderm. (G,H) Expression of Foxa2 is severely reduced (G) or absent (H) in mD2LIC-null mutant embryos. (I,J) Foxa2 expression is later reduced in anterior definitive endoderm in mD2LIC–/– embryos. (I) Wild-type embryo. (J) mD2LIC–/– embryo. Arrowheads mark anterior definitive endoderm. (K-P) TUNEL analysis of mD2LIC–/– embryos reveals no elevation of cell death in the node or its derivatives but apoptosis does occur in anterior definitive endoderm. (K-M) Little apoptosis is observed in the embryonic region of wild-type embryos at 7.5 dpc (K, lateral view; L, anterior view; M, distal view). In mD2LIC–/– embryos, no apoptosis is observed in the node or its derivatives (P) but substantial cell death occurs in the anterior definitive endoderm (arrowheads, N, lateral; O, anterior).

View larger version (93K): [in a new window]   Fig. 6. Analysis of mD2LIC–/– mutants at 8.5-9.0 dpc. (A,B) Expression of T is discontinuous and distorted in the notochord of embryos lacking mD2LIC. (A) Wild-type embryo; (B) mD2LIC–/– embryo. (C,D) Expression of Shh is also discontinuous in null mutant embryos (data not shown) and is absent in the ventral neural tube as seen in histological sections (black arrowheads; sections shown here are from the head region). (C) Wild-type embryo; (D) mD2LIC–/– embryo. (E,F) mD2LIC-null embryos lack expression of Foxa2 in notochord and ventral neural tube and expression is greatly reduced in definitive embryonic endoderm. (E) Wild-type embryo (ventral view); (F) mutant embryo (ventral view); Foxa2 expression is lacking in notochord and ventral neural tube (arrowhead), and there is a reduction of expression in definitive endoderm (arrow). (G-Q) Defective establishment and maintenance of the left-right axis in mD2LIC–/– mutants. Wild-type (G, distal view) asymmetric expression of nodal around the node is not observed in null mutants (H, distal view). At the early somite stage, left-lateral plate specific expression of nodal (I, posterior view) is either expressed bilaterally (J, ventral view), on the right (K, dorsal view, white arrowheads; black arrowhead indicates expression in the node), on the left or absent (data not shown). Expression of Leftb in the left side of the ventral neural tube (L, anterior view, arrowhead; broken white line indicates the anterior edge of the head folds) is absent in null mutants (M, dorsal view, arrowhead) and Ebaf expression is randomised (in this case showing bilateral expression). Left lateral plate Pitx2 specific expression (N, ventral view) is randomised in null mutants, appearing bilaterally in class I and II mutants (O,P, ventral view, arrowheads) or on the left (Q, left-hand embryo, ventral view, arrowhead) or on the right (Q, right-hand embryo, ventral view, arrow) in class III mutants. White lines indicate the boundary of the headfolds. (R,S) Lack of Fgf8 expression in the anterior neural ridge (ANR) of mD2LIC–/– embryos. (R) Wild-type embryo (ventral view). Arrowhead shows expression of Fgf8 in the ANR. (S) mD2LIC–/– mutant embryo (ventral view). Fgf8 expression does not occur the in ANR (arrowhead). (T-Y) TUNEL staining reveals altered apoptotic profiles during neural tube closure. Normal apoptosis in the region of the hindbrain (arrowheads; T, lateral view; U, dorsal view of trunk; V, dorsal view of head) is absent in mutant (arrowhead in W, lateral view; X,Y, dorsal view). In addition, ectopic cell death is observed along the dorsal midline in mutants (X, dorsal view of trunk, arrowhead), but does not occur in wild-type littermates (U). Cell death is also observed in the cephalic mesoderm surrounding the notochord in the region of the head (arrowheads, Y).

View larger version (37K): [in a new window]   Fig. 7. A model for the function of mD2LIC in the establishment of the body axes. (A) In wild-type embryos, mD2LIC is required for the formation of cilia in the node and for the correct morphology of ventral node cells (mottled grey circle). It is also necessary for the normal expression of Foxa2, Shh and T, and for the asymmetric expression of Nodal (dark blue), which leads to the induction of Nodal, Ebaf and Pitx2 in the left-hand-side of the embryo. The notochord (mottled rectangle below the node), also expresses Foxa2, Shh and T, while the adjacent ventral neural tube (light blue) expresses Foxa2 and Shh and the nodal signalling antagonist Leftb. These structures constitute the midline, which is thought to act as a barrier to maintain left-right character in the developing embryo. The anterior definitive endoderm (ADE, yellow) receives survival signals from node derivatives, including the axial mesendoderm and ventral neural tube, and the ADE in turn is thought to maintain an Fgf8-expressing signalling centre in the anterior neural ridge (ANR, purple). This is required for maintenance of the forebrain and anterior identity. (B) In mD2LIC–/– mutants ventral node cells do not form cilia and are flatter than their wild-type counterparts (solid grey circle). Expression of Foxa2, Shh and T is severely reduced or absent (depicted as faded text) and expression of Nodal is usually symmetrical. The compromised signalling properties of the organiser result in reduced expression of Shh, T and Foxa2 in the midline and consequently the absence of Foxa2, Shh and Leftb from the ventral neural tube. The bilaterally symmetrical expression of Nodal and the presumed loss of the midline barrier cause the nodal signalling pathway, normally active only in the left-hand side of the embryo, to be active on both sides. The defective axial mesendoderm does not emit survival signals to the ADE, and the Fgf8 signalling centre in the ANR is lost.

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