Fig. 4

Additional sites of hemorrhage in fibrinogen morphants.

Other sites of hemorrhage were visible in morpholino-injected larvae. Single cell embryos were injected with 1 nl of a mixture containing a final concentration of 0.5 mM of each of fga, fgb, and fgg splice blocking MOs, and then analyzed at 3 dpf by o-dianisidine staining. (a–c) Examples of morphants with intramuscular hemorrhage. Arrow shaped muscle segments are clearly highlighted by o-dianisidine staining, indicating hemorrhage. (d) Example of orbital hemorrhage. (e) Larvae were evaluated under a low power dissecting microscope and scored in the categories described. The 12 larvae with apparent muscle bleeds (as shown in a–c) also had hydrocephalus with or without ICH (intracranial hemorrhage). Control larvae displayed no abnormalities. (f) RT-PCR analysis of larvae after MO injection. Total mRNA was prepared from pools of 10 embryos or larvae that were uninjected (U), 1 day post injection (D1), or 3 days post injection (D3). RT-PCR was performed using primers in exons flanking the target splice donor sites. The quantity of parental cDNA product was diminished after injection, and altered splicing lead to additional products that were reduced (fga) or increased (fgb and fgg) in size, consistent with MO knockdown. elfa (elongation factor I alpha) was included as control for mRNA integrity. M, molecular weight marker with sizes in base pairs.