Wright CV et al. (1989), Interference with function of a homeobox gene i...

XB-ART-26454

Cell 1989 Oct 06;591:81-93. doi: 10.1016/0092-8674(89)90871-4.

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Interference with function of a homeobox gene in Xenopus embryos produces malformations of the anterior spinal cord.

Wright CV , Cho KW , Hardwicke J , Collins RH , De Robertis EM .

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XIHbox 1 is expressed in a narrow band across the cervical region of Xenopus embryos. The gene produces two related proteins: "long" and "short" XIHbox 1 homeodomain proteins. Injection of antibodies to the long XIHbox 1 protein into 1-cell embryos caused a phenotype in which the anterior spinal cord was morphologically transformed into a hindbrain-like structure. This alteration was restricted to the region normally expressing long XIHbox 1 protein. Injection of long protein mRNA disrupted segmentation and tissue organization without inhibiting cell proliferation. Injection of short protein mRNA into 1-cell embryos produced spinal cord malformations similar, but not identical, to those caused by the antibodies, suggesting antagonistic roles for long and short XIHbox 1 proteins. We immunostained tadpoles carrying extended hindbrains for N-CAM and consistently found defective organization of spinal nerves over the affected region.

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Species referenced: Xenopus laevis

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	Figure 1. An XlHbox I-Ab-Injected Tadpole with an Extended Hindbrain Line diagrams at the top of the figure indicate a control uninjected tadpole (I) and a long XlHbox lAb-injected tadpole with an extended hindbrain (II). The defect in the dorsal fin, by which this tadpole was originally identified, is shown in drawing II. A thick bar above each drawing indicates the anteroposterior band of expression of the long XlHbox 1 protein in Xenopus embryos. This region is also shaded in the CNS; note that in control tadpoles (I) it occupies spinal cord, but in the antibody-injected tadpole (II) it lies in a hindbrain-like structure complete with expanded fourth ventricle. (A-E) (control, tadpole I) and (A’-E’) (antibody-injected, tadpole II) show transverse sections taken at the levels indicated by the vertical arrows in drawings I and II. All sections were immunostained with long XlHbox IAb. The dorsal fin defect indicated in drawing II is visible in (C’) and (LY); compare with (C) and (D). Animals injected with the out-of-frame control antibody (see text and Table 1) are indistinguishable from the uninjected tadpole shown here. The position of the pronephros (PN) is shown. In (C) and (CT, the region of expression of long XlHbox 1 protein, which appears as dark nuclei, is indicated by an arrowhead. Note that posterior to the region of XlHbox 1 expression, the CNS adopts the morphology of normal spinal cord. Abbreviations: BA, branchial arch; DF, dorsal fin; EC, ependymal canal; HB, hindbrain; IV, the hindbrain cavity, called the fourth ventricle; WC, myotome; NO, notochord; SC, spinal cord. Bar = 50 pm.
	Figure 2. The Alterations in the Spinal Cord Extend the Fourth Ventricle into Regions Normally Occupied by Spinal Cord Higher magnifications of selected sections from the animals shown in Figure 1, giving prominence to the morphology of the CNS. (A-C) are from an uninjected tadpole, (A%‘) from a long XlHbox l-Ab-injected tadpole. The region where nuclei are positive (dark purple staining) for the long XlHbox 1 protein is indicated by an arrowhead in (B) and (B’). Abbreviations: EC, ependymal canal; HB, hindbrain; IV, hindbrain cavity called the fourth ventricle; WC, myotome; NO, notochord; SC, spinal cord. Bar = 50 urn.
	Figure 3. Injection of Long XlHbox l-Ab Produces Spinal Cord Defects over the Region of Expression of Long XlHbox 1 Protein Three 2.5-day-old tadpoles are presented in pairs of photographs (A, 8; C. D; E, F). (A) and (6) show a tadpole injected with an out-f-frame control antibody. (C)-(F) show two long XlHbox 1-Ab-injected tadpoles with phenotypic defects over the cervical spinal cord. The left-hand panel of each pair shows the embryo immunostained in whole mount with the spinal cord-specific XlHbox 6 antibody. XlHbox 6-positive nuclei in the spinal cord can be seen as dark brown dots over a uniform lighter-brown background. Over the region of spinal cord alteration in the antibody-injected tadpoles, the CNS resembles hindbrain, being closed dorsally by a thin roof and having a large cavity continuous with the fourth ventricle. Note that the position indicated by the arrow, which is within the region of the CNS affected by injecting long XlHbox lAb, expresses XlHbox 6 protein. Also note the position of the pronephros (PN) in relation to the normal spinal cord or morphologically transformed region of spinal cord, and the region of XIHbox 6 immunostaining. The right-hand panel of each pair shows a transverse section of the same embryo immunostained with long XlHbox 1Ab. The section was taken at the level indicated by the arrow in (A), (C), or(E), as appropriate. In the sections, the dark purple nuclear staining in the CNS shows that long XlHtxrx 1 protein is expressed in the cervical spinal cord of control tadpoles (B), but in the hindbrain-like regions of antibody-injected animals (D and F). Abbreviations: EC, ependymal canal: IV, hindbrain cavity called the fourth ventricle; SC, spinal cord.
	Figure 4. Phenotypic Effect of Microinjecting mRNA Encoding Long XlHbox 1 Protein (A) Synthetic XlHbox 1 mRNAs are efficiently translated in embryos. The embryo was injected at the 2-cell stage with long XlHbox 1 mRNA, incubated until blastula, and then immunostained in whole mount with antibodies against long XlHbox 1 protein. Large amounts of protein are found in all nuclei over half of the embryo, corresponding to cells that received the mRNA (intense dark staining in the upper region). At later stages, such as neurula and tailbud (not shown), cells containing long XlHbox 1 protein are found unilaterally through all three germ layers (although they are less stained at the tailbud stage). (B) Phenotypic effect of unilateral microinjection of long XlHbox 1 protein mRNA. This is a representative selection of four embryos exhibiting different degrees of the “bent” phenotype caused by unilateral injection of long protein mRNA at the 2-tell stage. The whole embryos are oriented anterior to the left, but are viewed as they come to rest naturally (mostly from a dorsal/lateral aspect). Whole-mount immunostaining shows that nuclei of all cells on the concave side (the side that received synthetic mRNA) contain long XlHbox 1 protein. (C) Horizontal longitudinal section (i.e., viewed from the dorsal aspect) of an uninjected control tadpole, stained with Hoechst 33258, showing normal bilateral ordered arrangement of myotome nuclei (arrowheads). Embryos microinjected at the 2-cell stage with mutant mRNA encoding the long XlHbox l/pro45 protein, which is incapable of sequence-specific DNA binding, are indistinguishable from uninjected tadpoles. ANT, anterior; POST, posterior. (D) One of the bent tadpoles shown in (8) was sectioned in the same orientation as (C) and stained with Hoechst 33256. Segmentation of the mesoderm and organization of the CNS in the left side of the embryo (bottom) are greatly disrupted. The uninjected side (top) appears relatively normal (compare the aligned myotome nuclei indicated by the arrowheads in [C] and [D]).
	Figure 5. Effect of Unilateral Injection at the Z-Cell Stage of mRNA Encoding the Short XlHbox 1 Protein The top two photographs (A, B) are of an embryo injected with mutant short XlHbox l/pro45 protein mRNA. Uninjected control tadpoles were identical. The bottom pair (C, D) corresponds to an embryo injected with short XlHbox 1 protein mANA. (A) and (C) show dorsal views of embryos immunostained in whole mount with long XlHbox 1Ab. (B) and (D) show transverse sections taken at the level of the arrows in (A) and (C), respectively, immunostained with long XlHbox I-Ab; positive nuclei appear black. Arrowheads in (C) and (D) indicate the reduced number of neurons expressing XlHbox 1 caused by producing short XlHbox 1 protein on this side of the embryo. In (8) and (D), XlHbox l-positive nuclei are present in the lateral plate mesoderm (Oliver et al., 1988; Wright et al., 1989a). Mydome cells do not express the XlHbox 1 protein. Abbreviations: ANT., anterior; POST., posterior; DF, dorsal fin; ENDO, endoderm; LPM, lateral plate mesoderm; MYO, myotome; NO, notochord; SC, spinal cord. Bar = 50 pm.
	Figure 6. Injection of mRNA Encoding Short XlHbox 1 Protein Changes the Morphology of Anterior Spinal Cord to That of Hindbrain (Top row) Control 3-day-old tadpole injected with mutant short XlHbox l/pro45 mRNA. Uninjected control tadpoles were identical. (Bottom row) Sibling 3-day-old tadpole injected with short XlHbox 1 protein mRNA. (A, A) Tadpoles were immunostained in whole mount with XlHbox 6 antibody and viewed dorsally. Specific nuclear staining is observed in the CNS, which runs horizontally from hindbrain to spinal cord, with XlHbox 6 staining beginning just ahead of arrow B or B’. XlHbox 6 is found in the spinal cord of the control tadpole, but in tadpoles showing cervical spinal cord malformations, XlHbox 6 is expressed in the new hindbrain-like region as well as the more posterior normal spinal cord. The unstained pronephri appear symmetrically as two darkened regions on each side of the control tadpole, but were accidentally removed together with the endoderm from the embryo injected with wild-type mRNA. The phenotype was analyzed further by serial sectioning and immunostaining with antibodies specific for XlHbox 1 and XlHbox 6. (B-D; S-D’) Transverse sections taken at the levels indicated by the arrows in (A) and (A)‘, respectively. All sections were immunostained with XlHbox 6 antibody, with positive nuclei appearing as black dots within the CNS. Comparison of the two rows of photographs shows that the affected cervical region resembles hindbrain rather than spinal cord, and expresses the spinal cord-specific XlHbox 6 protein over the normal anteroposterior limits. The alteration shown here extends more posteriorly than that seen in the long XlHbox l-Ab injection experiments. Abbreviations: HB, hindbrain; IV, hindbrain cavity called the fourth ventricle; SC, spinal cord.
	Figure 7. Defects in Spinal Nerves over the Affected Region of the Anterior Spinal Cord Tadpoles at 3 days of age were immunostained in whole mount with anti-N-CAM antibodies. (A) N-CAM staining pattern in a normal uninjected tadpole at low magnification. (6) High magnification of the same tadpole shown in (A). (C)A tadpole injected with long Xllibox I-Ah at the l-cell stage, carrying an extended hindbrain. Note the three roots entering the ganglion of the Ix/x cranial nerves and the meshwork of axons (mixing with axons coming from the vagus) that replaces the discrete bundles of spinal nerves seen in normal tadpoles. (D) A tadpole injected at the l-cell stage with mRNA encoding the short XlHbox 1 protein. Note the more extensive spinal cord malformation and the replacement of spinal nerves with a meshwork of axons that now occupies a much wider region than in the antibody-injected animal (C). Abbreviations: E, eye; AV, auditory vesicle; IX and X, ninth and tenth cranial nerves; SN, spinal nerves; M, diffuse meshwork of axons that would normally be discrete spinal nerves.