Development: Growing up to become a regenerative powerhouse

Summary:

Adult planarian flatworms respond to nearly any injury with a robust, whole-body regenerative response. Recent results demonstrate that planarians develop their regenerative prowess late in embryogenesis, following a late-emerging ability to reestablish anterior polarity.

Freshwater flatworms called planarians have captured the attention of scientists and naturalists for hundreds of years due to their robust regenerative capacity^{1, 2}. Adult planarians respond to injury with a suite of molecular and cellular responses that includes mobilization of adult, pluripotent stem cells called “neoblasts”^{3, 4}. Stem cells divide and differentiate to produce diverse progeny after injury, leading to full reestablishment of organ systems that include an intestine, muscles, neurons, epidermis, and an osmoregulatory protonephridia system⁵. New organs are created in the correct sizes and places due to the quick reestablishment of axial polarity after injury². While great progress has been made on understanding regeneration of adult planarians, far less is understood about how and when planarians acquire their regenerative abilities during embryogenesis.

Planarians develop within a brown, opaque cocoon that makes their embryonic development difficult to observe. Scientists spent over a hundred years completing meticulous studies on dissected planarian embryos, which together revealed a process that defies conventions of animal development^6-12. Through these studies, planarian embryos were determined to be ectolecithal, with initial blastomeres existing within a soup of yolk cells that eventually fuse into a syncytium. Blastomeres then enter a season of “blastomere anarchy,” dividing and detaching from one another to “wander” through the sea of yolk¹¹. Eventually these wandering blastomeres rise to a sufficient cell number that permits reorganization into a transient epidermis and embryonic pharynx; these structures allow the embryo to ingest the remaining yolk to provide building blocks for growth and to create an internal structure around which form cell layers and, eventually, the intestine¹¹. From mid-development onward, definitive organs are formed. New, columnar epidermal cells replace the transient epidermis, and a definitive pharynx and intestine arise. Similarly, neurons, muscle cells, and protonephridial cells are born and converge into organ primordia. As true organs begin to form, the embryo elongates. Prior to hatching from the cocoon, the embryonic body plan is very similar to that of the earliest juvenile stages. Juvenile planarians emerge with most body systems complete, with one major exception being reproductive structures that are elaborated later. During studies of planarian embryos, evidence emerged that regenerative ability was gained rather than retained over the course of embryogenesis. Several scientists noted that planarian embryos lacked the ability to regenerate until later stages^{7, 10}. In fact, until mid-embryogenesis, planarians fail to complete superficial wound healing and cannot survive outside of the cocoon⁷. Late in development, planarians gain regenerative capacity, acquiring adult-like regenerative abilities shortly before or after hatching.

The cellular and molecular reason(s) for regenerative failure in planarian embryos has remained unknown. In this issue, Booth and colleagues tackle this exciting, century-old developmental biology mystery¹³. One plausible reason for regenerative failure could be the lack of neoblasts capable of powering regeneration. Indeed, Davies and colleagues previously profiled dividing stem cells of the planarian Schmidtea mediterranea, revealing key molecular differences between the blastomeres of the embryo and adult neoblasts¹⁴. The differences between early blastomeres and late neoblasts have functional importance, as well. Mid-regeneration (stage 5), stem cells transplanted into an irradiated host can engraft but not divide¹⁴. By the latest stages of development, stem cells can engraft, divide, and also reestablish the stem cell population of the host¹⁴. Using a related planarian, Schmidtea polychroa, Booth and colleagues confirm the central role of stem cells in acquisition of regenerative ability, but also determine that the presence of adult-like stem cells is not sufficient to allow regeneration to proceed¹³. They also show that stem cells become heterogeneous and respond to injury at a stage prior to onset of full regenerative capacity.

Next, Booth and colleagues sought to determine the proximate cause of regenerative failure in planarian embryos. One important clue came from the observation that tail regeneration in embryos occurs at least a stage earlier than head regeneration¹³. The posterior bias of regenerative acquisition had also been noted prior in additional planarian species^{7, 10}. Therefore, Booth and colleagues hypothesized that while axial polarity is established relatively early during embryogenesis, the ability to reset axes might be acquired later. Indeed, embryos from nonregenerative stages failed to reestablish anteroposterior markers while embryos in regenerative stages succeeded in resetting polarity¹³. The posterior bias in embryonic regeneration could also be explained by the observation that planarian embryos gained the ability to reset the posterior pole a stage or more earlier than they succeeded at resetting the anterior pole¹³. In planarians, anterior polarity is inhibited by the Wnt pathway, through beta-catenin². Thus, Booth and colleagues confirmed that anterior polarity reset is critical for the acquisition of regenerative ability through an experiment in which they inhibited Wnt signaling in Stage 7 embryos and observed precocious head regeneration¹³. Taken together, this landmark study convincingly demonstrates that regeneration occurs only in planarian embryos with both regeneration-competent stem cells and an adult-like ability to rebuild all axes of polarity.

One question that remains is why planarian embryos fail at anterior polarity reset up until late in embryogenesis. Planarian polarity cues are produced in body wall muscle¹⁵ and anterior polarity cues are produced in longitudinal muscle fibers that depend on the transcription factor-encoding gene myoD¹⁶. Interestingly, myoD(RNAi) animals fail at both anterior and posterior regeneration, partly phenocopying the regenerative failure of embryonic planarians. Nonetheless, myoD transcript is expressed in planarian embryos prior to regeneration competence and myocytes are also evident early in development^{11, 13, 14}, suggesting that muscle cell presence is not sufficient for anterior reestablishment. However, muscle maturation and muscle fiber organization have not been fully assessed during planarian embryogenesis, raising the possibility that muscle physiology continues to change in late embryogenesis in ways that promote resilient polarity and regeneration. Further, the nervous system continues to mature in late embryogenesis, with brain condensation, axonal projections, and the nerve plexus all arising in Stage 7¹¹, so innervation of muscle could also be important for its anterior-promoting functions.

Another fascinating area of study will be comparative analyses of regeneration through the lens of embryogenesis. Regenerative abilities vary among flatworms and even between planarian species¹⁷. Strikingly, adults of several planarian species—Procotyla fluviatilis, Phagocata kawakatsui, and Dendrocoelum lacteum—fail at head regeneration. In all three species, beta-catenin(RNAi) rescues head regeneration^18-20, just as it does in the late S. polychroa embryo¹³. It will be fascinating to see if the mechanisms that drive failure of head regeneration overlap in non-regenerating species and non-regenerating developmental stages. More broadly, there are numerous animals in disparate animal phyla that complete whole-body regeneration, including acoels and Hydra, so the work in this issue sets a strong foundation for discovering and comparing how evolutionarily distant organisms establish their regenerative powers during embryogenesis.

Figure:

Planarians regenerate during a series of embryonic stages. Embryonic organs develop and are later replaced by definitive organs that include an intestine, a pharynx, epidermis, a central and peripheral nervous system, muscle, and protonephridia⁵. Sexual maturation occurs post-embryonically. The gradual exchange of embryonic tissues for definitive organs is represented in a color change from grays to blues. Yellow labels indicate distinct stages of organogenesis, while arrows indicate the timing with which stem cells adopt adult-like properties and embryos adopt adult-like regenerative abilities.