Discovery of transcription factors and other candidate regulators of neural crest development

Abstract

Neural crest cells migrate long distances and form divergent derivatives in vertebrate embryos. Despite previous efforts to identify genes up-regulated in neural crest populations, transcription factors have proved to be elusive due to relatively low expression levels and often transient expression. We screened newly induced neural crest cells for early target genes with the aim of identifying transcriptional regulators and other developmentally important genes. This yielded numerous candidate regulators, including 14 transcription factors, many of which were not previously associated with neural crest development. Quantitative real-time polymerase chain reaction confirmed up-regulation of several transcription factors in newly induced neural crest populations in vitro. In a secondary screen by in situ hybridization, we verified the expression of >100 genes in the neural crest. We note that several of the transcription factors and other genes from the screen are expressed in other migratory cell populations and have been implicated in diverse forms of cancer.

Figure 1

Neural crest gene expression: In situ hybridization was performed to confirm the expression of genes seen to be upregulated in macroarray screen. Selected genes from different categories are shown in dorsal view with anterior at the top. (A) chromatin: safB1; (B) cytoskeletal: paranemin; (C) ECM: lamin B2; (D) mitochondria: hexokinase 2; (E) mitosis/cell cycle: TD-60; (F) Nucleocytoplamic export: importin 9; (G) protein production/degradation: makorin; (H) receptors/downstream signaling: MHC-B; (I) rho pathway: kinectin; (J) RNA binding proteins: HNRPM; (K) secreted signals/signal production: PEBP; (L) transport: OSBP2; (M) miscellaneous: PGK; (N) unknown: chEST872f20.

Figure 2

Neural crest expression of and QPCR results for Snail 2 (slug). (A-D) Snail2 expression is seen specifically in premigratory (dorsal neural tube) and migratory neural crest. Shown are in situ hybridizations of 10 somite (A) and 13 somite (B-D) embryos in whole mount (A and B) and sections of whole mount stained embryos (C and D). Immunofluorescence for HNK-1 is in red (D). Arrowheads mark premigratatory neural crest, arrows mark migratory neural crest. Scale bars = X. (E) Results of conjugate QPCR showing a very highly significant difference between control and conjugate tissues (p = 0.00015).

Figure 3

Neural crest expression of and QPCR results for Elk-3. (A-D) Elk-3 expression is seen specifically in premigratory (dorsal neural tube) and migratory neural crest. Shown are in situ hybridizations of 10 somite (A) and 16 somite (B-D) embryos in whole mount (A and B) and sections of whole mount stained embryos (C and D). Immunofluorescence for HNK-1 is in red (D). Arrowheads mark premigratatory neural crest, arrows mark migratory neural crest.(E) QPCR results showing a highly significant difference between control and conjugated tissues (p = 0.005).

Figure 4

Neural crest expression of and QPCR results for GCNF. (A-D) GCNF expression is seen specifically in premigratory (dorsal neural tube) and migratory neural crest. Shown are in situ hybridizations of 12 somite (A) and 15 somite (B-D) embryos in whole mount (A and B) and sections of whole mount stained embryos (C and D). Immunofluorescence for HNK-1 is in red (D). Arrowheads mark premigratatory neural crest, arrows mark migratory neural crest. (E) results of QPCR showing a significant difference between control and conjugated tissues (p = 0.0384).

Figure 5

Neural crest expression of and QPCR results for GTF2E. (A-D) GTF2E expression is seen specifically in premigratory (dorsal neural tube) and migratory neural crest. Shown are in situ hybridizations of 10 somite (A) and 12 somite (B-D) embryos in whole mount (A and B) and sections of whole mount stained embryos (C and D). Immunofluorescence for HNK-1 is in red (D). Arrowheads mark premigratatory neural crest, arrows mark migratory neural crest. (E) results of QPCR showing a significant difference between control and conjugated tissues (p = 0.041).

Figure 6

Neural crest expression of and QPCR results for SREBP2. (A-D) SREBP2 expression is seen specifically in premigratory (dorsal neural tube) and migratory neural crest. Shown are in situ hybridizations of 9 somite (A) and 18 somite (B-D) embryos in whole mount (A and B) and sections of whole mount stained embryos (C and D). Immunofluorescence for HNK-1 is in red (D). Arrowheads mark premigratatory neural crest, arrows mark migratory neural crest. (E) results of QPCR showing a significant difference between control and conjugated tissues (p = 0.030).