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FIGURE 3 Zbtb14 is required for formation of posterior neural tissue and suppression of anterior neural development. (a–d) With or without β-gal mRNA, 35 ng of Control MO (a; Cont MO), 6mis Zbtb14 MOa (b), Zbtb14 MOa (c) or Zbtb14 MOa with 500 pg of 5′ del-zbtb14 mRNA (d) was injected unilaterally into 2- cell- stage embryos. Expression of ncam analyzed by whole- mount in situ hybridization is shown in purple and β- Gal is stained in red. The injected side was identified by either β- Gal staining or fluorescence signal (See Materials and Methods for details). In embryos injected with Zbtb14 MOa, expression of ncam on the injected side was severely reduced (arrowheads in c) and 5′ del-zbtb14 significantly rescued the reduction of ncam expression induced by Zbtb14 MOa (arrows in d). The injected side of the embryo is indicated by brackets. All panels show dorsal views with posterior to the top. (e) Summary of phenotypes obtained from several repeats of the experiments shown in (a–d). The degree of recovery in the ncam expression domains was scored and categorized into three types: ■, Severely; , Weakly; □, unchanged. Numbers of embryos per experimental group are indicated above the bars. (f–u) Together with β-gal mRNA, either Cont MO (35 ng, left columns) or Zbtb14 MOa (35 ng, right columns) was injected unilaterally into 2- cell- stage embryos. Expression of sox2 (early neural marker, f and g), ncam (late neural marker, h and i), n-tubulin (differentiated neuronal marker, j and k), epidermal keratin (E. keratin) (epidermal marker, l and m), myod (paraxial mesoderm marker, n and o), pax6 (neural plate and eye field marker, p and q), krox20 (hindbrain marker)/rx2a (eye field marker, r and s), and hoxb9 (spinal cord marker, t and u) genes analyzed by whole- mount in situ hybridization are shown in purple and β- Gal is stained in red. In embryos injected with Zbtb14 MOa, expression of ncam and n-tubulin on the injected side is reduced; however, expression of sox2 is not significantly reduced. The expression pattern of myod is not significantly affected by Zbtb14 MOa. Expression of rx2a on the injected side is expanded, whereas that of krox20 is reduced (s). Expression of hoxb9 is also decreased and shifted posteriorly (horizontal lines mark approximate anterior limits of hoxb9 expression; u). In accordance with these observations, the spinal cord expression of pax6 is suppressed (arrowheads), while the anterior expression domains of pax6 and rx2a are expanded toward the posterior and lateral sides (arrows) (q and s). The injected side of the embryo is indicated by brackets. All panels show dorsal views with posterior to the top, except for pax6 and krox20/rx2a, which show anterior (front) views with dorsal to the top.
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