Regeneration of the larval sea star nervous system by wounding induced respecification to the sox2 lineage

The ability to restore lost body parts following traumatic injury is a fascinating area of biology that challenges current understanding of the ontogeny of differentiation. The origin of new cells needed to regenerate lost tissue, and whether they are pluripotent stem cells, tissue-specific stem cells or have de- or trans- differentiated, remains one of the most important open questions in regeneration. Additionally, it is not clearly known whether developmental gene regulatory networks (GRNs) are reused to direct specification in these cells or whether regeneration specific networks are deployed. Echinoderms, including sea stars, have extensive ability for regeneration and have therefore been the subject of many thorough studies on the ultrastructural and molecular properties of cells needed for regeneration. However, the technologies for obtaining transgenic echinoderms are limited and tracking cells involved in regeneration, and thus identifying the cellular sources and potencies has proven challenging. In this study we develop new transgenic tools to follow the fate of populations of cells in the regenerating bipinnaria larva of the sea star Patira minaita. We show that the larval serotonergic nervous system can regenerate following decapitation. Using a BAC-transgenesis approach with photoconvertible fluorescent proteins, we show that expression of the pan ectodermal marker, sox2, is induced in previously sox2 minus cells at the wound site, even when cell division is inhibited. sox2+ cells give rise to new sox4+ neural precursors that then proceed along an embryonic neurogenesis pathway to reform the anterior nervous systems. sox2+ cells contribute to only neural and ectoderm lineages, indicating that these progenitors maintain their normal, embryonic lineage restriction. This indicates that sea star larval regeneration uses a combination of existing lineage restricted stem cells, as well as respecification of cells into neural lineages, and at least partial reuse of developmental GRNs to regenerate their nervous system.

Regeneration of the larval sea star nervous system by wounding induced respecification to the sox2 lineage

The ability to restore lost body parts following traumatic injury is a fascinating area of biology that challenges current understanding of the ontogeny of differentiation. The origin of new cells needed to regenerate lost tissue, and whether they are pluripotent stem cells, tissue-specific stem cells or have de- or trans- differentiated, remains one of the most important open questions in regeneration. Additionally, it is not clearly known whether developmental gene regulatory networks (GRNs) are reused to direct specification in these cells or whether regeneration specific networks are deployed. Echinoderms, including sea stars, have extensive ability for regeneration and have therefore been the subject of many thorough studies on the ultrastructural and molecular properties of cells needed for regeneration. However, the technologies for obtaining transgenic echinoderms are limited and tracking cells involved in regeneration, and thus identifying the cellular sources and potencies has proven challenging. In this study we develop new transgenic tools for cell tracking in the regenerating bipinnaria larva of the sea star Patira minaita. We show that the larval serotonergic nervous system can regenerate following decapitation. Using a BAC-transgenesis approach with photoconvertible fluorescent proteins, we show that expression of the pan ectodermal marker, sox2, is induced in previously sox2 minus cells at the wound site, even when cell division is inhibited. sox2+ cells give rise to new sox4+ neural precursors that then proceed along an embryonic neurogenesis pathway to reform the anterior nervous systems. sox2+ cells contribute to only neural and ectoderm lineages, indicating that these progenitors maintain their normal, embryonic lineage restriction. This indicates that sea star larval regeneration uses a combination of existing lineage-restricted stem cells, as well as respecification of cells into neural lineages, and at least partial reuse of developmental GRNs to regenerate their nervous system.

An inducible Cre mouse line to sparsely target nervous system cells, including Remak Schwann cells

ABSTRACTNerves of the peripheral nervous system contain two classes of Schwann cells– myelinating Schwann cells that ensheath large caliber axons in myelin, and Remak Schwann cells that surround smaller axons and do not myelinate. While tools exist for genetic targeting of myelinating Schwann cells, such reagents have been challenging to generate for the Remak population in part because many of the genes that mark this population in maturity are also robustly expressed in progenitors of all Schwann cells. To circumvent this challenge, we utilized BAC transgenesis to generate a mouse line expressing a tamoxifen-inducible Cre under the control of a Remak-expressed gene promoter (Egr1). However, as Egr1 is also an activity dependent gene expressed by some neurons, we flanked this Cre by flippase (Flpe) recognition target sites, and coinjected a BAC expressing Flpe under control of a pan-neuronal Snap25 promoter, to excise the Cre transgene from these neuronal cells. Genotyping and inheritance demonstrate that the two BACs co-integrated into a single locus, facilitating maintenance of the line. Anatomical studies following a cross to a reporter line show sparse tamoxifen-dependent recombination in Remak Schwann cells within the mature sciatic nerve. However, depletion of neuronal Cre activity by Flpe is partial, with some neurons and astrocytes also showing evidence of Cre reporter activity in the central nervous system. Thus, this mouse line will be useful in mosaic loss-of-function studies, lineage tracing studies following injury, and live cell imaging studies or other experiment benefiting from sparse labeling.

A Mammalian enhancer trap resource for discovering and manipulating neuronal cell types

There is a continuing need for driver strains to enable cell-type-specific manipulation in the nervous system. Each cell type expresses a unique set of genes, and recapitulating expression of marker genes by BAC transgenesis or knock-in has generated useful transgenic mouse lines. However, since genes are often expressed in many cell types, many of these lines have relatively broad expression patterns. We report an alternative transgenic approach capturing distal enhancers for more focused expression. We identified an enhancer trap probe often producing restricted reporter expression and developed efficient enhancer trap screening with the PiggyBac transposon. We established more than 200 lines and found many lines that label small subsets of neurons in brain substructures, including known and novel cell types. Images and other information about each line are available online (enhancertrap.bio.brandeis.edu).