Schmidtea mediterranea are flatworms with an incredible ability that lends an important role in the studies of developmental biology and medicine. These planarians can regenerate entire, fully developed adults from small tissue fragments, making them an ideal model organism for the study of body axis polarization and patterning. T.H. Morgan was first particularly fascinated by this process and performed several experiments to understand how polarity is reestablished (i.e. anterior-facing wound regenerates a head; posterior-facing wound regenerates a tail). Still, these regenerative processes are not yet fully understood; current studies led by several researchers in the field are rapidly providing new information to shed more light on this phenomenon.
The Wonders of Regeneration
Planarian with two heads on both ends
Figure 1. Formation of regeneration blastema after head amputation. Source: Life Illinois Newmark
After a planarian has been amputated, the wound is rapidly covered by a thin layer of epidermal cells. Undifferentiated cells called neoblasts are signaled to proliferate underneath the epithelium, producing an unpigmented structure known as the regeneration blastema (as seen in the figure above, merely 6 days after amputation). Neoblasts continue to propagate within the growing blastema, and within one week, differentiation of the missing structures occurs.
Figure 2. Morphogenesis of eyes. Source: Planiki Barcelona Planarian Lab
To ensure that these planarians regenerate the correct missing structures, several pathways are involved in regulating the reestablishment of polarity in these organisms. Much progress has been made in understanding these developmental pathways that control planarian regeneration. Particularly, Wnt/βcatenin signaling is essential to reestablishing the antero-posterior axis.
Reestablishing Antero-posterior (A-P) Axis
In Petersen and Reddien (2008), a S. mediterranea gene, Smed-βcatenin-1, was studied for its role in controlling A-P polarity. RNAi experiments were performed to examine its specific function during the reestablishment of A-P polarity.
When Smed-βcatenin-1(RNAi) organisms were amputated in several places (Fig. 3), they regenerated heads at all amputation sites, even from the posterior and side incisions. However, injection of Smed-βcatenin-1 caused polarity reversal in the mutants, indicating that the gene is needed in determining polarity during regeneration. This is because the Smed-βcatenin-1gene promotes posterior formation during A-P axis regeneration and inhibits anterior identity at posterior wounds.
Figure 3. Smed-βcatenin-1(RNAi) animals regenerated heads at all amputation sites, indicating that Smed-βcatenin-1 is involved in posterior formation during regeneration (Petersen and Reddien 2008)
Figure 4. Wnt signaling pathway. Source: Wikipedia
β-catenin proteins are effectors of Wnt signaling and act downstream of Wnt (Fig. 4). In the paper (Petersen and Reddien 2008), they found five Wnt-family genes in S. mediterranea (Smed-wntP-1; Smed-wntP-2; Smed-wntP-3; Smed-wnt2-1; and Smed-wnt11-1) expressed in distinct areas along the A-P axis through in-situ hybridization (Fig. 5).
- Smed-wntP-1: tail tip
- Smed-wntP-2: posterior and internally around pharynx
- Smed-wntP-3: anterior pharynx
- Smed-wnt2-1: laterally in anterior half
- Smed-wnt11-1: gradient from posterior
Wnt signaling is inhibited by secreted frizzle-related proteins. The sFRP gene (Smed-sFRP-1) is expressed at the anterior pole; under sFRP inhibiton, all Wnt genes except Smed-wnt2-1 are restricted to the posterior and expressed at posterior-facing wounds. In contrast, Smed-wnt2-1 is expressed near anterior-facing wounds. Following amputation, Smed-sFRP-1 was expressed at new anterior poles. These patterns indicate that A-P axis is regulated by anterior Wnt inhibition and posterior Wnt activation.
In Yazawa et al (2009), RNAi of Hedgehog and Patched in another planarian species, Dugesia japonica, showed that they are antagonists. RNAi of Patched resulted in “Janus-tails” phenotype in mutants (tails formed at anterior and posterior ends after amputation), suggesting that its role in head regeneration of planarians. RNAi of Hedgehog signaling component genes caused loss of tail identity and “Janus-heads” formation (two-headed mutant) because Hedgehog signaling is responsible for posterior specification in planarian regeneration.
Additionally, dsRNA was used to cause double gene knockdown of Patched and Wnt/β-catenin signaling, resulting in “Janus-heads” phenotype as well. RNAi of β-catenin results in the same phenotype, which still persists after simultaneous Patched and β-catenin RNAi, indicating Patched functions upstream of β-catenin signaling.
Wnt/βcatenin and Hedgehog/Patched
Petersen and Reddien (2008) show that Wnt/βcatenin signaling is necessary for posterior fate during regeneration. Smed-βcatenin-1 affects posterior regeneration decision by acting at posterior-facing wounds, and polarity is controlled by the A-P location of Wnt gene expression and signaling antagonists (sFRP-1).
Further studies (Yazawa et al, 2009) reveal that Hh signaling acts as a regulator of Wnt genes and specifies posterior identity by activating the transcription of Wnt genes at the posterior end. The posterior specification signal is further transmitted by β-catenin signaling in stem cells, which then gain a posterior-fate-specification through Wnt/ β-catenin signaling (Fig. 6).
Thus, Hh signaling is the activator of a key molecular switch for posterior specification, and Wnt/β-catenin signaling acts as the switch, playing a role in the reestablishment of AP polarity in planarians.
While both studies explain the pathways involved in posterior specification, they do not mention the mechanism that promotes anterior identity.
Smed-prep: TALE class homeobox gene
Felix and Aboobaker (2010) describe Smed-prep as a gene necessary for correct anterior specification during planarian regeneration. This TALE class homeobox gene is expressed at high levels in the anterior portion of whole planarians.
RNAi experiments show the vital role of Smed-prep in anterior reestablishment. Smed-prep(RNAi) resulted in all worms having either a cyclops phenotype (fusion of two eyes) or no eyes at all with no defects in posterior blastema formation. These mutants also exhibited brain loss during anterior regeneration, but not during lateral regeneration or homeostasis in whole worms. Double knockdown experiments with the S. mediterranea ortholog of nou-darake (when knocked down induces ectopic brain formation) show that Smed-prep defines an anterior fated compartment where stem cells are allowed to differentiate into the brain, but the gene is not required directly for brain differentiation.
Expression of markers of different anterior fated cells are greatly reduced or lost in the absence of Smed-prep; Wnt signaling inhibition without Smed-prep is not enough to cause anterior structures to form during regeneration. These results indicate that Smed-prep is the first gene discovered that is involved in promoting anterior fate and correct patterning.
Reestablishing the Dorsal-Ventral (DV) Axis
BMP/ADMP Regulatory Circuit
The BMP pathway is required to specify and maintain dorsal identity during planarian regeneration (Gavino and Reddien, 2011). ADMP (anti-dorsalizing morphogenetic protein) and BMPs show complementary expression patterns that identify the dorsal and ventral midlines as well as the lateral, dorsal-ventral axis of planarians.
BMP/ADMP works as a regulatory circuit. Smed-admp(RNAi) caused regeneration of indented heads and tails, indicating that admp is required for regeneration of tissues at lateral edges at the midpoint between dorsal and ventral poles. When these RNAi mutants were exposed to a low dose of bmp4 dsRNA, they were more sensitive to small decreases in Bmp signaling level during DV regeneration and responded by becoming ventralized near wound sites. Smed-admp promotes Smed-bmp4 expression; admp signaling is required to maintain the appropriate level and broad spatial distribution of bmp4 expression during adult tissue maintenance and growth. Smed-admp is expressed at ventral pole and laterally in adult Schmidtea mediterranea, spatially opposing the dorsal-pole domain of Smed-bmp4. Smed-admp promotes Smed-bmp4 expression, which inhibits Smed-admp expression, regulating circuit that buffers against perturbations of Bmp signaling.
Loss of function of the ligand bmp or intracellular elements, smad1 or smad4-1 results in partial ventralization, where an ectopic CNS and mouth opening differentiate dorsally and expression of some dorsal markers disappear and dorsal epithelia cilia develop a ventral-like pattern (Molina et al, 2011). But silencing of noggins (well-known antagonists of BMP signaling) yields complementary dorsalizing phenotypes in which dorsal markers and differentiating eyes develop in the anterior ventral region.
BMP/ADMP Circuit Regulation
Silencing of noggin-like gene 8 (nlg8) yields similar phenotypes of bmp knockdowns with dorsal CNS differentiation, but not thickening, DV border duplication or disappearance of dorsal markers, and ventralization of dorsal cilia. bmp or smad1 loss-of-function results in posterior-to-anterior differentiation of ectopic dorsal nerve cords, but nlg8 silencing ectopic CNS appears preferentially in anterior region and seems to connect with a dorsal expansion of ventral cephalic ganglia. These results lead to the conclusion that BMP/ADMP circuit is regulated by noggin and noggin-like genes.
Felix, D., and Aboobaker, A. (2010). The TALE Class Homeobox Gene Smed-prep Defines the Anterior Compartment for Head Regeneration. PLoS Genetics 6(4).
Gaviño, Michael A., and Peter W. Reddien. (2011). A Bmp/Admp Regulatory Circuit Controls Maintenance and Regeneration of Dorsal-Ventral Polarity in Planarians. Current Biology 21(4), 294–299.
Molina, M.D., E. Saló, and F. Cebrià. (2011). Organizing the DV Axis During Planarian Regeneration. Communicative & integrative biology, 4(4), 498.
Petersen, C. and Reddien, W. (2008). Smed-Bcatenin-1 is Required for Anteroposterior Blastema Polarity in Planarian Regeneration. Science, 319, 327-329.
Yazawa et al. (2009). Planarian Hedgehog/Patched establishes anterior-posterior polarity by regulating Wnt signaling. PNAS, 106(52), 22329-22334.