Regeneration in the era of functional genomics and gene network analysis

Abstract

What gives an organism the ability to regrow tissues and to recover function where another organism fails is the central problem of regenerative biology. The challenge is to describe the mechanisms of regeneration at the molecular level, delivering detailed insights into the many components that are cross-regulated. In other words, a broad, yet deep dissection of the system-wide network of molecular interactions is needed. Functional genomics has been used to elucidate gene regulatory networks (GRNs) in developing tissues, which, like regeneration, are complex systems. Therefore, we reason that the GRN approach, aided by next generation technologies, can also be applied to study the molecular mechanisms underlying the complex functions of regeneration. We ask what characteristics a model system must have to support a GRN analysis. Our discussion focuses on regeneration in the central nervous system, where loss of function has particularly devastating consequences for an organism. We examine a cohort of cells conserved across all vertebrates, the reticulospinal (RS) neurons, which lend themselves well to experimental manipulations. In the lamprey, a jawless vertebrate, there are giant RS neurons whose large size and ability to regenerate make them particularly suited for a GRN analysis. Adding to their value, a distinct subset of lamprey RS neurons reproducibly fail to regenerate, presenting an opportunity for side-by-side comparison of gene networks that promote or inhibit regeneration. Thus, determining the GRN for regeneration in RS neurons will provide a mechanistic understanding of the fundamental cues that lead to success or failure to regenerate.

Figure 1

Overview of gene regulatory network (GRN) approach. The middle column depicts the general work flow for the GRN approach with landmark data sets shown in the Appendix. The left column highlights the scientific questions addressed by each component; the right column identifies the corresponding steps as labeled in text. To sum up, the GRN approach follows the classic parameters of observation, controlled experimentation, and hypothesis testing. During the observation phase, expressed regulatory factors are identified by deep transcriptome sequencing, and their spatial and temporal expression patterns are determined at high resolution (Appendix Part A). Next, an individual regulatory gene is perturbed and transcript levels for all other genes quantitatively assessed by high-throughput methods (see text and Appendix Part B); this process is then repeated for all expressed genes, while parallel ChIP-seq assays provide a complementary data set to identify candidate cis-acting genomic elements. All data sets are then cross-referenced to generate predictions of the network of interactions. These predictions are validated in mechanistic detail by reporter assays; the dynamic series of regulatory steps is then tested by network re-engineering (Appendix Part C).

Figure 2

Time course of behavioral recovery in lamprey after spinal cord transection. The lamprey spontaneously recovers swimming movements after complete spinal cord transection in a stereotypic manner over the course of 12 weeks. The numbers on the y-axis correspond to reproducible stages of recovery and are adapted from Oliphint et al. (2010). Briefly, a score of 0 indicates that the animal can move its head rostral to the transection site and is paralyzed below it. A score of 1 indicates that the animal can curl into a C- or S-shape. A score of 2 indicates the ability to accomplish abnormal, brief bouts of self-initiated swimming. A 3 indicates the ability to sustain more persistent, but still abnormal, bouts of swimming, including abnormal body contractions and difficulty righting. Finally, a score of 4 indicates that the animal can swim consistently, persistently, in a manner that appears as if uninjured. Data are derived from the mean ± sem of 5–6 animals (Bloom, O, unpubl. data).

Figure 3

Regeneration in the lamprey nervous system. (A) Diagram of lamprey CNS. Giant reticulospinal (RS) neurons in midbrain and hindbrain project directly to spinal cord. The typical spinal cord injury paradigm is a complete spinal cord transection, which axotomizes all descending neurons, including giant RS neurons. (B) A Nissl-stained lamprey brain showing the locations of all identified giant RS neurons. Giant RS neurons include the large Müller cells in the mesencephalic (M), isthmic (I), and bulbar (B) brain regions, as well as the Mauthner (Mth) cell. (C) A Nissl-stained cross-section of the lamprey spinal cord showing locations of giant RS axons in the ventromedial tract. The axons of the Mauthner (Mth) neurons are located more dorsolaterally. Dorsal (D) and ventral (V) orientations are indicated. CC = central canal. (D) After transection, some giant RS neurons regenerate reliably, while others do not. Here, “poor regenerators” (red) are defined as those neurons that regenerate <50% of the time (see Jacobs et al., 1997). (E) A schematic of the distal lamprey spinal cord at 11 weeks post-transection. Mauthner axons are rarely observed because they are poor regenerators. Only about 50% of the giant RS neurons regenerated.