Fig. 3. The normal preferential extension of interstitial branches toward their TZ is altered by ectopic domains of ephrin-B1 consistent with a repellent action. (A) Quantification scheme. The tectum was divided into three domains: medial of the TZ, within the LM extent of the TZ, and lateral of the TZ. Branches in each of these bins were scored as either directed laterally or medially, and as within or outside a transfection domain. The blue and green arrowheads represent the relative strength and direction of the branching preference. A Directional Coefficient (DC) was calculated by subtracting the percentage of branches directed laterally from the percentage of branches directed medially. A positive DC indicates a preference to branch medially, whereas a negative DC indicates a preference to branch laterally. (B,C) In control E11-E14 chicks most branches formed along temporal RGC axons, outside the LM extent of the TZ, extend towards the TZ. At the LM position of the TZ branches show no preference in orientation. The temporal control cases (B) included 14 normal, non-transfected tecta (n=499 branches). The nasal-ventral control cases (C) included 3 RCAS-eGFP-transfected tecta, 1 tectum electroporated with RCAS-ephrin-B1-IRES-eGFP, but in which no eGFP reporter labeling was detected, and 7 normal non-transfected tecta (n=399 branches). (D) In chicks transfected with RCAS-ephrin-B1-IRES-eGFP (n=11 tecta, 700 branches), quantitation of branch directionality, irrespective of their relationship to ectopic domains of ephrin-B1, shows a disruption in normal directionality and a bias to extend laterally. (E) Branches from RCAS-ephrin-B1-IRES-eGFP-transfected cases that were located within an ectopic domain of ephrin-B1 expression were directed laterally, regardless of position (n=11 tecta, 386 branches). (F) Branches in RCAS-ephrin-B1-IRES-eGFP transfected cases that were located outside an ectopic domain of ephrin-B1 exhibited normal branching preferences toward their TZ (n=11 tecta, 314 branches). Statistical tests of significance of quantitation of directional extension of interstitial branches: Nasal-ventral controls (C): in the lateral bin, more branches were directed medially than laterally, Student's paired t-test, P<0.01; at the LM location of the nascent TZ, there is no difference in branch directionality, P=0.87; in the medial bin, more branches are directed laterally than medially, P<0.04. Temporal controls (B) show the same branch directionality as the nasal-ventral controls (C): lateral bins, P=0.92; TZ bins, P=0.80; medial bins, P=0.84 (² test). Test of significance for all interstitial branches in ephrin-B1 transfected tecta (D) compared to nasal-ventral controls (C): lateral bins, P<<0.001 (² test); TZ and medial bins are not significantly different. Test of significance in ephrin-B1-transfected tecta for branch directionality within ectopic domains of ephrin-B1 (E) compared to nasal-ventral controls (C): lateral bins, P<<0.001; TZ bins, P<0.04; medial bins, P=0.367 (² test). Test of significance in ephrin-B1-transfected tecta for branch directionality within ectopic domains of ephrin-B1 (E) versus outside of the domains (F): lateral bins, P<<0.001; TZ bins, P<0.04; medial bins, P<0.1 (² test). Test of significance in ephrin-B1 transfected tecta for branch directionality outside of ectopic domains of ephrin-B1 (F) compared to nasal-ventral controls (C): lateral bins, P=0.863; TZ bins, P=0.85; medial bins, P=0.52 (² test).

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