Downloaded from genesdev.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press To proliferate or to die: role of Id3 in cell cycle progression and survival of neural crest progenitors Yun Kee and Marianne Bronner-Fraser1 Division of Biology, California Institute of Technology, Pasadena, California 91125, USA The neural crest is a unique population of mitotically active, multipotent progenitors that arise at the vertebrate neural plate border. Here, we show that the helix–loop–helix transcriptional regulator Id3 has a novel role in cell cycle progression and survival of neural crest progenitors in Xenopus. Id3 is localized at the neural plate border during gastrulation and neurulation, overlapping the domain of neural crest induction. Morpholino oligonucleotide-mediated depletion of Id3 results in the absence of neural crest precursors and a resultant loss of neural crest derivatives. This appears to be mediated by cell cycle inhibition followed by cell death of the neural crest progenitor pool, rather than a cell fate switch. Conversely, overexpression of Id3 increases cell proliferation and results in expansion of the neural crest domain. Our data suggest that Id3 functions by a novel mechanism, independent of cell fate determination, to mediate the decision of neural crest precursors to proliferate or die. [Keywords: Xenopus; Id3; neural crest; cell cycle; survival] Received September 1, 2004; revised version accepted January 19, 2005. The neural crest is an embryonic cell population that origi- et al. 2003), c-Myc (Bellmeyer et al. 2003), and Msx1 nates from the lateral edges of the neural plate during (Tribulo et al. 2003) have been identified in Xenopus nervous system formation. As the neural plate folds and as transcriptional regulators of neural crest specifica- transforms into the neural tube, neural crest cells emi- ion, the molecular understanding of how these cells grate from the forming central nervous system, prolifer- arise and the molecular network linking these regula- ate extensively, and migrate to numerous destinations. ors of neural crest formation still remains to be established. They then differentiate into diverse derivatives ranging Members of the Id (inhibitor of DNA binding or in- from neurons and glia of the peripheral nervous system hibitor of differentiation) gene family possess a helix– to cartilage of the face, smooth muscle, and melanocytes loop–helix domain that is required for dimerization (Groves and Bronner-Fraser 1999; LaBonne and Bronner- and negatively influences the ability of tissue-specific Fraser 1999; Le Douarin and Kalcheim 1999; Knecht and basic helix–loop–helix (bHLH) transcription factors to Bronner-Fraser 2002; Gammill and Bronner-Fraser 2003). bind to DNA (Norton et al. 1998; Norton 2000; Yokota Although many studies have focused on induction of 2001; Tzeng 2003). The Drosophila ortholog of Id, extra- the neural crest precursor pool, the molecular signals macrochaetae (emc), antagonizes Daughterless and ach- and mechanisms underlying early events such as cell aete-scute bHLH proteins involved in sex deterination proliferation and survival of the neural crest are not yet and neurogenesis, acting as a negative regulator of differ- understood. In Xenopus, neural crest induction requires entiation (Ellis et al. 1990; Garrell and Modolell 1990). at least two signals including Wnt/FGF signaling and The Id genes have highly overlapping expresion patterns inhibition of BMP signaling (Saint-Jeannet et al. 1997; in mice (Evans and O’Brien 1993; Jen et al. 1997; Andres- LaBonne and Bronner-Fraser 1998; Marchant et al. 1998). Barquin et al. 2000), making analysis of their loss-of- Although several transcription factors such as Snail function phenotype difficult because of functional re- (Essex et al. 1993), Slug (LaBonne and Bronner-Fraser dundancy. Targeted gene disruption of Id1, Id2, or Id3 in 2000; Mayor et al. 2000), Sox9 (Spokony et al. 2002), mice yields mutant animals that develop normally and Sox10 (Aoki et al. 2003; Honore et al. 2003), AP2␣ (Luo are viable, with major defects appearing post-natally in the immune system (Lyden et al. 1999). Double knock- 1Corresponding author. out mice of Id1 and Id3 survive to adulthood; however, E-MAIL [email protected]; FAX (626) 395-7717. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ neuroblasts in the brain prematurely differentiate, impli- gad.1257405. cating Id genes as positive regulators of cell cycle control 744 GENES & DEVELOPMENT 19:744–755 © 2005 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/05; www.genesdev.org Downloaded from genesdev.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press Id3 in NCC proliferation and survival of neural precursors (Lyden et al. 1999). These mice also Results exhibit vascular malformations in the forebrain and de- Id3 is expressed in premigratory and migratory neural fects in the branching and sprouting of blood vessels into crest cells during Xenopus embryogenesis the neuroectoderm. Chick Id2 is localized in the cranial neural folds and subsequently in a subpopulation of mi- Neural crest development requires proper expression of grating cranial neural crest cells, and overexpression ex- regulatory molecules in neural crest precursor cells at periments in the chick suggest that Id2 may play a role in appropriate times. Although the expression patterns of neural crest development in this species (Martinsen and Id2, Id3, and Id4 have previously been examined in Bronner-Fraser 1998). However, loss-of-function analysis Xenopus embryos (Wilson and Mohun 1995; Zhang et al. was not performed due to technical difficulties in the 1995; Liu and Harland 2003), no Id gene was identified in chick. early neural crest progenitors in Xenopus. Our in situ Id genes have been implicated in the regulation of di- hybridization revealed that Xenopus Id3, but not other Id verse cellular events such as controlling cell cycle, pro- genes, was expressed in neural crest progenitors. We liferation, differentiation, or apoptosis of different cell then performed a detailed analysis of the expression pat- types in several in vitro mammalian cell line models tern of Id3 as a function of time from gastrulation (Tzeng 2003). However, the mechanisms that mediate through the early stages of neural crest migration. At the their effects in developing tissues in vivo are not well beginning of gastrulation, Id3 was expressed broadly understood. Xenopus embryos offer several advantages throughout the non-neural ectoderm in the animal half for functional analysis of genes involved in neural crest of the embryo. As gastrulation proceeded, Id3 expression development because it is possible to focally inject was down-regulated above the dorsal lip (Fig. 1A). During mRNAs and small interfering constructs into specific initial stages of patterning of the prospective neural tissues for both gain- and loss-of-function studies. Here, plate, it was down-regulated in the newly formed neural we report that Xenopus Id3 is the only Id expressed in plate in a pattern emanating from the organizer toward newly formed and migrating neural crest; furtherore, the dorsal and anterior region of the embryos (Fig. 1B). At morpholino antisense oligonucleotide-mediated knock- early neurula stages, the expression was maintained at down of Id3 protein causes depletion of neural crest the neural plate border where neural crest precursors lie, precursors and their derivatives. This loss apparently oc- but down-regulated elsewhere (Fig. 1C,D). In addition to curs via cell cycle inhibition followed by cell death of the neural folds, prominent expression was noted in two the precursor population rather than via a cell fate stripes at the mid–hindbrain boundary and hindbrain switch. Our results demonstrate an essential role of Id3 level (Fig. 1D,E). in mediating neural crest cell proliferation and cell sur- To verify that Id3 was, indeed, expressed in the neural vival. crest, we performed double in situ hybridization with Figure 1. Id3 is expressed in premigratory and migratory neural crest cells. (A) Id3 ex- pression at the gastrula stage (stage 10.5). Id3 expression is seen in the ectoderm of the animal half of the embryo and starts to decrease in the region above the dorsal lip (arrow). (B–D) Dorsal views of embryos at stages 12.5–15 (anterior toward left). Id3 expression is progressively down-regulated through late-gastrula and early-neurula stages and maintained at the neural plate border. Arrows indicate blastopore (B), and neural fold (C,D). (D) The arrowhead indi- cates the hindbrain region. (E) Anterior lat eral view of Id3 expression at stage 15. Id3 is clearly expressed in the anterior neural plate (arrow), midbrain and hindbrain re- gions in two stripes, and at the neural plate border including the premigratory neural crest domain (arrowhead). (F) Double in situ hybridization with Id3 and FoxD3 RNA. The foxD3 expression (purple) overlaps with the Id3 expression (light blue) in the neural crest domain (arrowhead). The Id3-expressing region is slightly larger than the foxD3-expressing neural crest domain. (G) Dorsal view of Id3 expression at stage 21. Id3 is expressed in migrating cranial neural crest cells (arrow) and the trunk neural fold region (arrowhead). (H) Dorsal view of Id3 expression at stage 23. Id3 is expressed in the dorsal neural tube at cranial and trunk levels (arrowhead)aswellas in the optic placode (arrow). (I) Section of an early-neurula-stage embryo showing Id3 expression in the superficial layer (arrow) at the border of neuronal and non-neuronal ectoderm. (J,K) Section of an embryo showing migrating neural crest cells (arrow) coming out of the cranial dorsal neural tube at stage 22. Id3 expression in the dorsal neural tube (J) is magnified in K. Id3 is also expressed in the optic placode (arrowhead). (NT) Neural tube. (L) Id3 expression in the trunk dorsal neural tube and neural crest cells (arrow) coming out of the dorsal neural tube as well as in the ectoderm above it.