Development Genes and Evolution

, Volume 222, Issue 3, pp 139–151 | Cite as

Analysis of snail genes in the crustacean Parhyale hawaiensis: insight into snail gene family evolution

  • Roberta L. Hannibal
  • Alivia L. Price
  • Ronald J. Parchem
  • Nipam H. Patel
Original Article


The transcriptional repressor snail was first discovered in Drosophila melanogaster, where it initially plays a role in gastrulation and mesoderm formation, and later plays a role in neurogenesis. Among arthropods, this role of snail appears to be conserved in the insects Tribolium and Anopheles gambiae, but not in the chelicerates Cupiennius salei and Achaearanea tepidariorum, the myriapod Glomeris marginata, or the Branchiopod crustacean Daphnia magna. These data imply that within arthropoda, snail acquired its role in gastrulation and mesoderm formation in the insect lineage. However, crustaceans are a diverse group with several major taxa, making analysis of more crustaceans necessary to potentially understand the ancestral role of snail in Pancrustacea (crustaceans + insects) and thus in the ancestor of insects as well. To address these questions, we examined the snail family in the Malacostracan crustacean Parhyale hawaiensis. We found three snail homologs, Ph-snail1, Ph-snail2 and Ph-snail3, and one scratch homolog, Ph-scratch. Parhyale snail genes are expressed after gastrulation, during germband formation and elongation. Ph-snail1, Ph-snail2, and Ph-snail3 are expressed in distinct patterns in the neuroectoderm. Ph-snail1 is the only Parhyale snail gene expressed in the mesoderm, where its expression cycles in the mesodermal stem cells, called mesoteloblasts. The mesoteloblasts go through a series of cycles, where each cycle is composed of a migration phase and a division phase. Ph-snail1 is expressed during the migration phase, but not during the division phase. We found that as each mesoteloblast division produces one segment’s worth of mesoderm, Ph-snail1 expression is linked to both the cell cycle and the segmental production of mesoderm.


Parhyale hawaiensis snail scratch Arthropod Crustacean 



We thank other members of the Patel lab for the following: Cristina Grande for help with the phylogenetic analysis, Francis Poulin for screening the Parhyale BAC library for Ph-sna1, and Crystal Chaw and Angela Kaczmarczyk for insightful comments. For the tdTomato-Moesin construct, we thank Roger Tsien for the tdTomato, Dan Kiehart for Drosophila Moesin, E. Jay Rehm for the Parhyale hawiensis EF1α promoter, and Paul Liu for creating the tdTomato-Moesin construct.

Supplementary material

427_2012_396_Fig8_ESM.jpg (353 kb)
Supplementary Figure 1

Protein alignment used for phylogenetic analysis. Abbreviations: Am Apis mellifera, Dm Drosophila melanogaster, Dp Daphnia pulex, Hs Homo sapiens, Lg Lottia gigantea, Nv Nematostella vectensis, Ph Parhyale hawaiensis, Tc Tribolium castaneum, Sp Strongylocentrotus purpuratus. Sequence sources same as in Fig. 1 (JPEG 352 kb)

427_2012_396_MOESM1_ESM.tif (1.1 mb)
High resolution image (TIFF 1161 kb)
427_2012_396_Fig9_ESM.jpg (30 kb)
Supplementary Figure 2

Ph-sna1, Ph-sna2, Ph-sna3, and Ph-scratch BAC Schematic. BAC DNA represented by black, red, and orange lines; BAC DNA matching cDNA represented by red and orange line(s) (snail and scratch homologs, respectively). Arrows point to exons; orientation of BACs is 5′ to 3′ with respect to gene(s) in that BAC. 289E23 and 185A16 both contain the Ph-sna1 cDNA, revealing the absence of introns in this gene. In addition, 185A16 contains the Ph-sna3 cDNA. There are two exons for Ph-sna3 contained in this BAC. The Ph-sna2 BAC, 268N10, contains the Ph-sna2 cDNA. There are two exons for Ph-sna2 contained in this BAC. The Ph-scratch BAC, 026E11, contains the Ph-scratch cDNA. There are two exons for Ph-scratch contained in this BAC. Scale bar = 10,000 base pairs (JPEG 29 kb)

427_2012_396_MOESM2_ESM.tif (279 kb)
High resolution image (TIFF 278 kb)
427_2012_396_Fig10_ESM.jpg (223 kb)
Supplementary Figure 3

Zinc-finger alignment of the snail superfamily. Protein alignment of the region containing the five conserved zinc fingers amongst members of the snail superfamily. Zinc fingers are boxed in red for Snail homologs and orange for Scratch homologs. Abbreviations: Am Apis mellifera, Dm Drosophila melanogaster, Dp Daphnia pulex, Hs Homo sapiens, Lg Lottia gigantea, Nv Nematostella vectensis, Ph Parhyale hawaiensis, Tc Tribolium castaneum, Sp Strongylocentrotus purpuratus. Sequence sources same as in Fig. 1 (JPEG 223 kb)

427_2012_396_MOESM3_ESM.tif (1.7 mb)
High resolution image (TIFF 1752 kb)
427_2012_396_MOESM4_ESM.doc (49 kb)
Supplementary Figure 4 Annotated Snail superfamily amino acid sequences. Zinc fingers highlighted in yellow, SNAG domain highlighted in green, NT box highlighted in cyan, and CtBP interaction motif highlighted in magenta. Abbreviations: Am Apis mellifera, At Achaearanea tepidariorum, Cs Cupiennius salei, Dm Drosophila melanogaster, Dp Daphnia pulex, Gm Glomeris marginata, Hs Homo sapiens, Lg Lottia gigantean, Nv Nematostella vectensis, Ph Parhyale hawaiensis, Tc Tribolium castaneum, Sp Strongylocentrotus purpuratus. Sequence sources same as in Fig. 1, except for the addition of the following sequence sources from GenBank: At-sna AB167392; Cs-sna AJ571697; Gm-sna DQ408593 (DOC 49 kb)
427_2012_396_MOESM5_ESM.m4v (2.6 mb)
Supplementary Movie 1 Mesoteloblast migration and division. Mesoteloblasts migrate posteriorly in the embryo; during this migration, they undergo a series of divisions to produce the mesoblasts. Timelapse movie of an embryo where the mesoderm progenitors mr and ml were injected with DsRed-NLS, which marks cell nuclei (for details, see the “Materials and methods” section). Ventral view; anterior to the top. Mesoteloblasts are the large, most posterior cells (arrowheads at beginning of movie). Their progeny, the mesoblasts, are the small, more anterior cells that will form one row of mesoderm per segment (brackets indicate segments T2-T4 at end of movie). Cells anterior to the second thoracic segment (T2) are head and trunk segmental mesoderm derived from mr and ml formed through either divisions of a combination of mesoteloblasts and mesoteloblast precursors, or a mesotelobalsts-independent mechanism (Price and Patel 2008). While not completely synchronous, the mesoteloblasts are generally migrating and dividing at the same time (Price and Patel 2008). In this movie, divisions are seen when the punctate DsRed-NLS becomes diffuse as the nuclear envelope breaks down, followed by the production of a new mesoblast (yellow arrowheads at beginning of movie indicate mesoteloblasts that have just finished dividing) (M4V 2615 kb) (8.9 mb)
Supplementary Movie 2 Mesoteloblast display dynamic cell shape changes. Timelapse movie of a close up of the right side of an embryo where the mesoderm progenitor mr was injected with tdTomato-Moesin DNA to mark cell membranes (for details, see the “Materials and methods” section). Ventral view; anterior is to the top. Mesoteloblasts are the large, most posterior cells, and their progeny, the mesoblasts, are the small, more anterior cells. The most lateral mesoteloblast, M4, and progeny are to the left, the rest of the mesoteloblasts and their progeny are to the right. When the mesoteloblasts are not dividing, they produce filopodia that contact both the other mesodermal cells and the ectoderm. Arrowhead marks the most prominent filopodia of M4. Right before and during division, the mesoteloblasts retracts their filopodia. Asterisk marks M4 right before and during division (MOV 9097 kb)


  1. Alberga A, Boulay JL, Kempe E, Dennefeld C, Haenlin M (1991) The snail gene required for mesoderm formation in Drosophila is expressed dynamically in derivatives of all three germ layers. Development 111:983–992PubMedGoogle Scholar
  2. Ashraf SI, Hu X, Roote J, Ip YT (1999) The mesoderm determinant Snail collaborates with related zinc-finger proteins to control Drosophila neurogenesis. EMBO J 18:6426–6438PubMedCrossRefGoogle Scholar
  3. Barrallo-Gimeno A, Nieto MA (2005) The Snail genes as inducers of cell movement and survival: implications in development and cancer. Development 132:3151–3161PubMedCrossRefGoogle Scholar
  4. Barrallo-Gimeno A, Nieto MA (2009) Evolutionary history of the Snail/Scratch superfamily. Trends Gen 25:248–252CrossRefGoogle Scholar
  5. Browne WE, Price AL, Gerberding M, Patel NH (2005) Stages of embryonic development in the amphipod crustacean, Parhyale hawaiensis. Genesis 42:124–149Google Scholar
  6. Dale JK, Pascale M, Chal J, Vilhais-Neto G, Maroto M, Johnson T, Jayasinghe S, Trainor P, Herrmann B, Pourquie O (2006) Oscillations of the Snail genes in the presomitic mesoderm coordinate segmental patterning and morphogenesis in vertebrate somitogenesis. Dev Cell 10:355–366PubMedCrossRefGoogle Scholar
  7. Fuse N, Hirose S, Hayashi S (1996) Determination of wing cell fate by the escargot and snail genes in Drosophila. Development 122:1059–1067PubMedGoogle Scholar
  8. Gerberding M, Browne WE, Patel NH (2002) Cell lineage analysis of the amphipod crustacean Parhyale hawaiensis reveals an early restriction of cell fates. Development 129:5789–5801PubMedCrossRefGoogle Scholar
  9. Goltsev Y, Fuse N, Frasch M, Zinzen RP, Lanzaro G, Levine M (2007) Evolution of the dorsal-ventral patterning network in the mosquito, Anopheles gambiae. Development 134:2415–2424PubMedCrossRefGoogle Scholar
  10. Grau Y, Carteret C, Simpson P (1984) Mutations and chromosomal rearrangements affecting the expression of snail, a gene involved in embryonic patterning in Drosophila melanogaster. Genetics 108:347–360PubMedGoogle Scholar
  11. Hannibal RL, Price AL, Patel NH (2012) The functional relationship between ectodermal and mesodermal segmentation in the crustacean, Parhyale hawaiensis. Dev Biol 361:427–438PubMedCrossRefGoogle Scholar
  12. Hemavathy K, Ashraf SI, Ip YT (2000) Snail/slug family of repressors: slowly going into the fast lane of development and cancer. Gene 257:1–12PubMedCrossRefGoogle Scholar
  13. Ip YT, Park RE, Kosman D, Bier E, Levine M (1992) The dorsal gradient morphogen regulates stripes of rhomboid expression in the presumptive neuroectoderm of the Drosophila embryo. Genes Dev 6:1728–1739PubMedCrossRefGoogle Scholar
  14. Kasai Y, Nambu JR, Lieberman PM, Crews ST (1992) Dorsal-ventral patterning in Drosophila: DNA binding of snail protein to the single-minded gene. Proc Natl Acad Sci USA 89:3414–3418PubMedCrossRefGoogle Scholar
  15. Kerner P, Hung J, Behague J, Le Gouar M, Balavoine G, Vervoort M (2009) Insights into the evolution of the snail superfamily from metazoan wide molecular phylogenies and expression data in annelids. BMC Evol Biol 9:94PubMedCrossRefGoogle Scholar
  16. Koerner TJ, Hill JE, Myers AM, Tzagoloff A (1991) High-expression vectors with multiple cloning sites for construction of trpE fusion genes: pATH vectors. Methods Enzymol 194:477–490PubMedCrossRefGoogle Scholar
  17. Kosman D, Ip YT, Levine M, Arora K (1991) Establishment of the mesoderm-neuroectoderm boundary in the Drosophila embryo. Science 254:118–122PubMedCrossRefGoogle Scholar
  18. Leptin M (1991) twist and snail as positive and negative regulators during Drosophila mesoderm development. Genes Dev 5:1568–1576PubMedCrossRefGoogle Scholar
  19. Manzanares M, Locascio A, Nieto MA (2001) The increasing complexity of the Snail gene superfamily in metazoan evolution. Trends Gen 17:178–181CrossRefGoogle Scholar
  20. Marin F, Nieto MA (2006) The expression of Scratch genes in the developing and adult brain. Dev Dyn 235:2586–2591PubMedCrossRefGoogle Scholar
  21. Nakakura EK, Watkins DN, Schuebel KE, Sriuranpong V, Borges MW, Nelkin BD, Ball DW (2001) Mammalian Scratch: a neural-specific Snail family transcriptional repressor. Proc Natl Acad Sci USA 98:4010–4015PubMedCrossRefGoogle Scholar
  22. Nieto MA (2002) The snail superfamily of zinc-finger transcription factors. Nat Rev Mol Cell Biol 3:155–166PubMedCrossRefGoogle Scholar
  23. Nusslein-Volhard C, Wieschaus E, Kluding H (1984) Mutations affecting the pattern of the larval cuticle in Drosophila melanogaster. Wilhelm Roux Arch Dev Biol 193:267–282CrossRefGoogle Scholar
  24. Palmeirim I, Henrique D, Ish-Horowicz D, Pourquié O (1997) Avian hairy gene expression identifies a molecular clock linked to vertebrate segmentation and somitogenesis. Cell 91:639–648PubMedCrossRefGoogle Scholar
  25. Parchem RJ, Poulin F, Stuart AB, Amemiya CT, Patel NH (2010) BAC library for the amphipod crustacean, Parhyale hawaiensis. Genomics 95:261–267PubMedCrossRefGoogle Scholar
  26. Patel NH, Ball EE, Goodman CS (1992) Changing role of even-skipped during the evolution of insect pattern formation. Nature 357:339–342PubMedCrossRefGoogle Scholar
  27. Peel AD, Telford MJ, Akam M (2006) The evolution of hexapod engrailed-family genes: evidence for conservation and concerted evolution. Proc Roy Soc B Biol Sci 273:1733–1742CrossRefGoogle Scholar
  28. Pioro HL, Stollewerk A (2006) The expression pattern of genes involved in early neurogenesis suggests distinct and conserved functions in the diplopod Glomeris marginata. Dev Gene Evol 216:417–430CrossRefGoogle Scholar
  29. Pourquié O (2011) Vertebrate segmentation: from cyclic gene networks to scoliosis. Cell 145:650–663PubMedCrossRefGoogle Scholar
  30. Price AL, Modrell MS, Hannibal RL, Patel NH (2010) Mesoderm and ectoderm lineages in the crustacean Parhyale hawaiensis display intra-germ layer compensation. Dev Biol 341:256–266Google Scholar
  31. Price AL, Patel NH (2008) Investigating divergent mechanisms of mesoderm development in arthropods: the expression of Ph-twist and Ph-mef2 in Parhyale hawaiensis. J Exp Zool B Mol Dev Evol 310B:24–40CrossRefGoogle Scholar
  32. Regier JC, Shultz JW, Zwick A, Hussey A, Ball B, Wetzer R, Martin JW, Cunningham CW (2010) Arthropod relationships revealed by phylogenomic analysis of nuclear protein-coding sequences. Nature 463:1079–1083PubMedCrossRefGoogle Scholar
  33. Rehm EJ, Hannibal RL, Chaw RC, Vargas-Vila MA, Patel NH (2009) The crustacean Parhyale hawaiensis: a new model for arthropod development. In: Crotty DA, Gann A (eds) Emerging model organisms: a laboratory manual, vol 1. Cold Spring Harbor Laboratory Press, New York, pp 373–404Google Scholar
  34. Roark M, Sturtevant MA, Emery J, Vaessin H, Grell E, Bier E (1995) scratch, a pan-neural gene encoding a zinc finger protein related to snail, promotes neuronal development. Genes Dev 9:2384–2398PubMedCrossRefGoogle Scholar
  35. Sander K (1976) Specification of the basic body pattern in insect embryogenesis. Adv Insect Physiol 12:125–238CrossRefGoogle Scholar
  36. Sommer RJ, Tautz D (1994) Expression patterns of twist and snail in Tribolium (Coleoptera) suggest a homologous formation of mesoderm in long and short germ band insects. Dev Gene 15:32–37CrossRefGoogle Scholar
  37. Ungerer P, Eriksoon BJ, Stollewerk A (2011) Neurogenesis in the water flea Daphnia magna suggests different mechanisms of neuroblast formation in insects and crustaceans. Dev Biol 357:42–52PubMedCrossRefGoogle Scholar
  38. Vargas-Vila MA, Hannibal RL, Parchem RJ, Liu PZ, Patel NH (2010) A prominent requirement for single-minded and the ventral midline in the dorsoventral axis of the crustacean Pahyale hawaiensis. Development 137:3469–3476PubMedCrossRefGoogle Scholar
  39. Vega S, Morales AV, Ocaña OH, Valdés F, Fabregat I, Nieto MA (2004) Snail blocks the cell cycle and confers resistance to cell death. Genes Dev 18:1131–1143PubMedCrossRefGoogle Scholar
  40. Weller M, Tautz D (2003) Prospero and Snail expression during spider neurogenesis. Dev Gene Evo 11:554–566CrossRefGoogle Scholar
  41. Whiteley M, Noguchi PD, Sensabaugh SM, Odenwald WF, Kassis JA (1992) The Drosophila gene escargot encodes a zinc finger motif found in snail-related genes. Mech Dev 36:117–127PubMedCrossRefGoogle Scholar
  42. Yamazaki K, Akiyama-Oda Y, Oda H (2005) Expression patterns of a twist-related gene in embryos of the spider Achaearanea tepidariorum reveal divergent aspects of mesoderm development in the fly and spider. Zoo Sci 22:177–185CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Roberta L. Hannibal
    • 1
    • 2
    • 4
  • Alivia L. Price
    • 1
    • 2
    • 3
  • Ronald J. Parchem
    • 1
    • 2
  • Nipam H. Patel
    • 1
    • 2
  1. 1.Department of Molecular and Cell BiologyUniversity of CaliforniaBerkeleyUSA
  2. 2.Department of Integrative BiologyUniversity of CaliforniaBerkeleyUSA
  3. 3.Department of Molecular Genetics and Cell Biology, Committee on Developmental BiologyUniversity of ChicagoChicagoUSA
  4. 4.Department of GeneticsStanford University School of MedicineStanfordUSA

Personalised recommendations