Combined lineage mapping and fate specification profiling with NLOM-OCM using sub-10-fs pulses

摘要

We have developed a combined NLOM-OCM method using ultrashort sub-10-fs pulses to study cell lineages and their gene expression profiles in zebrafish. First, time-lapse NLOM is used to capture embryo morphology (broadly excited autofluorescence) and cell lineage dynamics (eGFP reporter). The embryo is then fixed and an in situ hybridization performed, depositing NBT/BCIP precipitate where a gene of interest is actively expressed. Combined NLOM-OCM is then used to capture the gene expression pattern with 3-D resolution and these two data sets acquired from the same embryo are merged using morphological landmarks. We have used this approach to study the dynamics of the wntl lineage at the midbrain-hindbrain boundary (MHB) in normal and in fgf8a(ace) morphant embryos. We show that with fgf8a knockdown, the MHB constriction begins to form but subsequent failure of the constriction causes the incorporation of a transient cerebellar structure into caudal tectum. Concomitantly, this morphological distortion in the dorsal MHB causes anterior displacement in a ventral subpopulation of the wntl lineage at the MHB. NLOM-OCM confirms the displaced wntl MHB lineage stops expressing the wntl reporter, and with further experiments we can investigate markers such as wnt4 or asclla, which have been shown to be expanded caudally in ace mutants, to understand the transformed molecular fate of this displaced tissue. We conclude this approach of co-registering dynamic lineage tracing and in situ hybridization data sets using morphological context will help shed light on developmental mechanisms by integrating established analysis techniques at the morphological, cellular, and molecular levels.