BACKGROUND: Grainyhead genes encode a family of transcription factors that are well conserved from fly to human. The three mammalian homologues, Grainyhead-like-1, -2, and -3 are expressed in various ectodermal and endodermal cell types during embryonic development and in adult skin. Gene targeting in mice has demonstrated functional roles for Grhl1 and Grhl3 in epidermal integrity and wound healing ability of the epidermis, which appear functionally related to the role of Drosophila grainyhead in production and healing of the epidermal cuticle. Importantly, targeted null mice for Grhl3 also display NTDs, comprising severe spina bifida as well as occasional exencephaly. The chromosomal location of Grhl3 and the finding of NTDs in null embryos suggested that Grhl3 could be allelic with the mouse mutant curly tail, a well known model for NTDs. Expression analysis and transgenic rescue suggest that curly tail is a hypomorphic allele of Grhl3. The functional role and downstream mediators of Grhl3 in neural tube closure are largely unknown. However, the developmental and cellular basis of NTDs in curly tail mutants is well established, involving a proliferation defect in the hindgut endoderm. CONCLUSIONS: On this basis, it is possible that Grhl3 has a direct regulatory function in cell proliferation in the hindgut endoderm. Identification of the transcriptional targets of Grhl3 will serve not only to further our understanding of the mechanisms of mammalian neural tube closure, but also to identify potential molecular factors involved in the pathogenesis of NTDs in human. Birth Defects Research (Part A), 2008. © 2008 Wiley-Liss, Inc.

GRAINYHEAD (GRH) AND THE GRH GENE FAMILY

GRH was first identified in Drosophila as a protein that can bind regulatory elements and modulate transcription of developmentally regulated genes, including the DOPA decarboxylase (Ddc) gene and ultrabithorax (Biggin and Tjian, 1988; Bray et al., 1988, 1989; Dynlacht et al., 1989). Functional studies of this transcription factor, initially named Elf-1 (element l-binding activity) or neuronal transcription factor 1, showed that it was identical with Grainyhead (grh), a previously identified embryonic lethal locus in Drosophila (Nüsslein-Volhard et al., 1984; Bray and Kafatos, 1991). GRH has been shown to be capable of both activation and repression of transcription (Attardi et al., 1993; Huang et al., 1995; Liaw et al., 1995; Hayashi et al., 1999). Indeed, the protein structure includes domains involved in DNA binding and dimerization located in the C-terminus of the protein, and an activation domain located in the N-terminus (Attardi et al., 1993; Uv et al., 1994). Dimerization results in stabilization of the DNA-protein complex (Uv et al., 1994).

GRH was the first member to be identified of a family of transcription factors that are evolutionary conserved from fly to human (Bray and Kafatos, 1991; Wilanowski et al., 2002; Venkatesan et al., 2003; Ting et al., 2003b). Phylogenetic analysis subdivides these genes into two main groupings, depending whether they are more related to grh, or to a second Drosophila gene, CP2 (Wilanowski et al., 2002; Ting et al., 2003b). For grh, three mammalian homologues have been identified: grainyhead-like-1 (Grhl1, also known as Mammalian Grainyhead or LBP-32), Grhl2 (Brother of Mammalian Grainyhead), and Grhl3 (Sister Of Mammalian Grainyhead [SOM] or Grainyhead-like Epithelial Transactivator) (Wilanowski et al., 2002; Kudryavtseva et al., 2003; Ting et al., 2003b). This group of mammalian Grainyhead-like genes encodes proteins of similar size that are highly homologous in the N-terminus as well as in the DNA binding and C-terminus dimerization domains (Wilanowski et al., 2002; Ting et al., 2003b). A second group of genes is more closely related to CP2 and consists of CP-2, LBP-1a, and LBP-9 (Ting et al., 2003b).

In this review we will focus principally on the mammalian Grhl3 gene, and its particular role in neural tube closure.

Grhl3 AND MAMMALIAN NEURAL TUBE CLOSURE

Grhl3 Function Is Required for Spinal Neural Tube Closure

The function of Grhl3 has been investigated by the analysis of gene-targeted lines of mice, carrying deletions of parts of exon 2 and exon 3, encoding the transactivation domain (Ting et al., 2003a), or exons 4–7 (Yu et al., 2006). These targeted alleles are both predicted to be functionally null, and resultant phenotypes appear to be very similar (although a direct side-by-side comparison has not been reported). Among various defects, homozygous Grhl3 null embryos exhibit fully penetrant spina bifida, an NTD that results from a failure of the neural folds to fuse in the caudal region of the developing embryo (Copp et al., 2003). In addition, closure of the cranial folds also fails in a proportion of embryos, resulting in exencephaly, the developmental forerunner of anencephaly. The frequency of exencephaly was 2 and 14% in the two targeted alleles (Ting et al., 2003a; Yu et al., 2006), which may be a consequence of the different targeting strategies or, possibly, difference in the genetic background of the resultant mouse line.

The high degree of sequence similarity between mouse GRH-like proteins and the ability of Grhl3 to form homodimers and heterodimers with Grhl1 or Grhl2 (Kudryavtseva et al., 2003) raises the question of whether these proteins also play a role in neural tube closure. Targeted null mutants for Grhl1 exhibit skin and hair abnormalities but not NTDs (Wilanowski et al., 2008). Moreover, complementation studies do not provide evidence for a genetic interaction between Grhl1 and Grhl3: Grhl1^−/−;Grhl3^+/− mice resemble Grhl1 null animals, whereas double null mutants develop NTDs that are indistinguishable from the Grhl3^−/− phenotype (Wilanowski et al., 2008). Reports of the Grhl2 knockout phenotype are awaited.

Diminished Grhl3 Expression as the Cause of NTDs in the Curly Tail Mouse

The Grhl3 gene was localized to mouse chromosome 4 by fluorescent in situ hybridization on metaphase chromosomes, radiation hybrid mapping (Kudryavtseva et al., 2003), and subsequently by genomic sequencing. This location corresponds to the region to which the spontaneous mutation, curly tail, was mapped in linkage studies (Neumann et al., 1994; Brouns et al., 2005). Curly tail is one of the most well-established mouse models for NTDs. Homozygous mutant ct/ct embryos develop tail flexion defects with lumbosacral spina bifida (10–20%) or tail flexion defects alone (40–50%), and with exencephaly in 1–5% of embryos. The remaining 40–60% of ct/ct embryos are straight tailed and apparently normal (Greene and Copp, 1997; van Straaten and Copp, 2001). Despite having been first described in the 1950s (Gruneberg, 1954), the causative curly tail genetic mutation was long unidentified, in part owing to the difficulties imposed on mapping studies by the partial penetrance of the defects and the significant effect of modifier genes. Therefore, the overlapping chromosomal locations and similar phenotypes implicated Grhl3 as a candidate gene for curly tail, although a coding region mutation has not been found (Ting et al., 2003a; Brouns et al., 2005; Gustavsson et al., 2007). Genetic complementation studies between Grhl3 null mutants and curly tail mice show a genetic interaction, with compound mutant offspring exhibiting NTDs that resemble the curly tail phenotype in both penetrance and expressivity (Ting et al., 2003a). However, genetic interaction cannot be taken as proof of allelism because the ct mutation also interacts with the splotch mutation in Pax3 and the loop tail mutation in Vangl2 (Estibeiro et al., 1993; Stiefel et al., 2007).

The developmental basis of spinal NTDs in curly tail embryos has been well characterized (Fig. 1), which provided an opportunity to evaluate Grhl3 as a potential causative gene, and may in turn shed light on the mechanism underlying spina bifida in Grhl3 knockout embryos. Tail defects and spina bifida in ct/ct embryos result from delayed closure of the posterior neuropore, the final region of the neural tube to close in the caudal region of the embryo (Copp, 1985). Delayed closure is caused by excessive ventral curvature of the caudal region of the embryo, which contains the neuropore (Brook et al., 1991). The angle of curvature is dictated by the relative longitudinal growth of dorsal and ventral tissues, and abnormally reduced cell proliferation rates are detected in ventral tissues, the notochord and hindgut endoderm of affected ct/ct embryos (Copp et al., 1988a). That this under-proliferation leads directly to NTDs is indicated by the finding that minimization of the growth imbalance, either through overall growth retardation or stimulation of hindgut proliferation, can normalize curvature and decrease the frequency of delayed posterior neuropore closure (Copp et al., 1988b; Cogram et al., 2004).

Details are in the caption following the image — **Figure 1**
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Summary of the mechanism underlying development of spinal NTDs in *curly tail* embryos. The sequence of events leading to delay or failure of posterior neuropore closure is indicated by solid arrows. The influence of modifier genes has not yet been established, but they could act upstream or downstream of *Grhl3.* Closure can be normalized by stimulation of hindgut proliferation (e.g., by inositol) or overall growth retardation, which ameliorates the growth imbalance between tissues.

Overall, these findings imply that, should aberrant expression of Grhl3 contribute to curly tail NTDs, then altered expression would be detectable in the caudal region of the embryo prior to failure of posterior neuropore closure, at embryonic day (E) 10.5. The pattern of expression of Grhl3 during embryogenesis is rather dynamic, with mRNA mainly localized to developing epithelia (Ting et al., 2003a; Auden et al., 2006; Gustavsson et al., 2007). In the context of spinal neurulation, Grhl3 transcripts are detected in the neuroepithelium of the posterior neuropore at E9.5. However, during the period encompassing posterior neuropore closure, at E10.5 (25–31 somites), expression is down-regulated in the neural folds and becomes evident in the hindgut endoderm (Fig. 2), the site of the cellular proliferation defect in ct/ct embryos (Gustavsson et al., 2007). Quantitative real-time RT-PCR analysis showed that, compared to genetically matched wild-type embryos, ct/ct embryos exhibited diminished abundance of Grhl3 transcripts in the caudal region, containing hindgut endoderm, at the stage of posterior neuropore closure (Gustavsson et al., 2007). These findings support the idea that insufficient expression of Grhl3 in the hindgut endoderm located ventrally to the posterior neuropore could be causally related to spina bifida in curly tail embryos. As a further test of this hypothesis, Grhl3 was over-expressed in ct/ct embryos by means of BAC (Bacterial Artificial Chromosome) transgenesis. A BAC from the RPCI24 library (C57BL/6J), containing the complete Grhl3 gene including putative regulatory regions, was injected in pronuclei of fertilized curly tail oocytes. Litters containing transgenic offspring were genotyped for the BAC and phenotypically scored for spina bifida and curled tail. Among transgenic litters, neither tail flexion defects nor spina bifida were found to occur in BAC-positive embryos or pups, whereas ct/ct littermates that did not carry the BAC developed NTDs at the expected frequency (Gustavsson et al., 2007).

The complete rescue of spinal NTDs by reinstatement of Grhl3 expression in ct/ct embryos strongly suggests that ct is a hypomorphic allele of Grhl3. It will be interesting to know whether Grhl3 null embryos exhibit enhanced ventral curvature of the caudal region, preceding failure of posterior neuropore closure, as would be predicted from studies in curly tail (Brook et al., 1991). Spina bifida in Grhl3 knockout mice occurs at 100% penetrance and lesions are more extensive than in curly tail mice: spina bifida in Grhl3 knockout embryos involves thoracic, lumbar, and sacral regions, whereas some ct/ct mice have large lumbosacral lesions while others have relatively small asymptomatic lesions (van Straaten and Copp, 2001; Ting et al., 2003a). We assume that the relatively milder phenotype of ct/ct embryos compared to Grhl3^−/− embryos is a consequence of the residual level of Grhl3 transcripts in ct, which encode wild-type protein.

Although it is apparent that diminished expression of Grhl3 predisposes ct/ct embryos to spinal NTDs, the reason for the development of defects in some embryos, whereas others are apparently unaffected, is not yet understood at the molecular level. For example, we do not detect a consistent difference in Grhl3 expression levels between ct/ct embryos undergoing delayed or normal posterior neuropore closure, even within litters (Gustavsson et al., 2007). Thus, susceptibility, as determined by proliferation rate in the hindgut, may also be influenced by other downstream or interacting factors. In fact, NTDs in curly tail appear exquisitely sensitive to both genetic modifiers (see below) and environmental factors, including retinoic acid and inositol (Chen et al., 1994, 1995; Greene and Copp, 1997). Inositol supplementation reduces the frequency of spina bifida by normalization of posterior neuropore closure (Fig. 1), an effect that depends on the activity of specific isoforms of protein kinase C (Greene and Copp, 1997; Cogram et al., 2002, 2004). In contrast, there is an apparent lack of a protective effect of inositol in Grhl3 null embryos (Ting et al., 2003a), which is presumably due to the greater severity and penetrance of defects.

What Is the Nature of the Curly Tail Mutation?

The Grhl3 gene encompasses 16 known exons spanning approximately 31 kb of genomic DNA, and encoding a 603 amino acid protein (Ting et al., 2003a). Because there are no coding mutations in the Grhl3 gene in curly tail, a PCR-based approach was undertaken to identify alternatively transcribed exons in the mouse Grhl3 gene. Indeed, there are at least three distinct transcripts in humans (Ting et al., 2003b). 5′- and 3′-RACE was performed on first strand cDNA derived from neurulation stage wild-type mouse embryos (E8.5–10.5) (Gustavsson et al., 2007). In addition, in order to identify coding sequences close to or within the Grhl3 gene region, the genomic sequence including the complete Grhl3 gene (BAC RPCI23-321M9; NCBI accession number AL670720) was analyzed for known genes, transcripts, or predicted genes using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) and GRAIL (http://compbio.ornl.gov/Grail-1.3/) analyses. Primers specific for putative upstream exons were designed and used for PCR on cDNA from both wild-type and curly tail embryos to screen for potential alternative transcripts. All of these approaches failed to identify any alternative transcripts in mouse embryos, and the curly tail cDNA was indistinguishable from wild-type (Gustavsson et al., 2007).

As an initial attempt to identify possible regulatory elements in the Grhl3 gene, prediction of upstream promoter elements was performed using the PROSCAN promoter recognition program (http://www-bimas.cit.nih.gov/molbio/proscan/). This revealed three predicted promoter elements within 8 kb upstream of the Grhl3 ATG, although no mutations were identified in sequence analysis of curly tail DNA. In parallel, sequencing of curly tail genomic DNA was performed to screen for genetic alterations in regulatory sequences not recognized by PROSCAN. Six genetic alterations were identified in a 30 kb region upstream of the Grhl3 ATG. However, only one of these changes, a single nucleotide substitution, had not previously been identified as either a single nucleotide polymorphism or a di- or tetranucleotide repeat difference (Gustavsson et al., 2007). This nucleotide (C/T) substitution is located more than 20 kb upstream of the Grhl3 ATG. We found that the 500 bp region containing this nucleotide change has enhancer activity, as shown by luciferase assay, suggesting that the nucleotide substitution may be located in a putative regulatory element. Further studies are needed to investigate the impact of this putative mutation on the Grhl3 gene expression in wild-type and curly tail spinal neural tube.

The reduced penetrance and variable expressivity of defects in homozygous curly tail mice, when bred onto different genetic backgrounds, triggered efforts to identify modifier genes (Neumann et al., 1994; Letts et al., 1995). Assuming the curly tail locus is allelic with Grhl3, modifier genes may include genes that are important for Grhl3 expression, downstream targets of Grhl3, and/or proteins that interact with Grhl3. Three modifier loci for curly tail have been mapped using recombinant inbred strains (Neumann et al., 1994; Letts et al., 1995). In particular, a modifier locus at chromosome 17 has been mapped, with a highly significant multipoint lod-score. Homozygosity at the chromosome 17 locus was shown to be important for spina bifida to occur in curly tail embryos (Letts et al., 1995).

Cranial NTDs Occur with Incomplete Penetrance in Grh-like 3 Mutant Mice

The occurrence of exencephaly in a proportion of Grhl3 knockout and curly tail embryos is currently unexplained (van Straaten and Copp, 2001; Ting et al., 2003a; Yu et al., 2006). This defect may be related to the expression of Grhl3 in the ventral forebrain during cranial neurulation (Fig. 2A) (Gustavsson et al., 2007), however, the function of Grhl3 in cranial neural tube closure is not well understood. Interestingly, Grhl3 was first identified on the basis of its physical interaction with another transcription factor, Lmo4, mutants of which also display exencephaly (Kudryavtseva et al., 2003; Hahm et al., 2004). Double knockouts for Grhl3 and Lmo4 exhibit an increased frequency of exencephaly, indicating a genetic interaction between Grhl3 and Lmo4 (Yu et al., 2006). LMO4 is a transcriptional coregulator, thought to function as a molecular adaptor for the assembly of multiprotein complexes (Sugihara et al., 1998; Kudryavtseva et al., 2003). Another Lmo4 interacting protein, Deformed Epidermal Autoregulatory Factor-1, is also required for cranial neural tube closure in mice (Hahm et al., 2004). Hence, Grhl3 may be involved in cranial neural tube closure via its participation in a complex of transcription factors and regulatory interactions.

A further link between Grhl3 and exencephaly comes from recent studies in which Grhl3 was found to be a direct transcriptional target gene of p53 during nerve growth factor-induced differentiation of PC12 cells (Brynczka et al., 2007). Exencephaly occurs in a proportion of p53 null mice (Sah et al., 1995), and it was suggested that the requirement for p53 function in neural tube closure could be mediated, at least in part, through regulation of Grhl3 expression (Brynczka et al., 2007).

GRH GENES HAVE DIVERSE DEVELOPMENTAL FUNCTIONS

At present, the molecular mechanism by which loss of Grhl3 function leads to NTDs is poorly understood and key downstream targets have not yet been identified. In contrast, the role of grh genes in a range of other processes (Table 1), including epidermal barrier formation, have been more extensively characterized at the molecular level. Moreover, clues to Grhl3 function may be obtained from a consideration of model systems such as Drosophila, which do not undergo neural tube formation, but are likely to share conserved regulatory pathways.

Table 1. Grainyhead-Like Genes Associated with Defects

Species	Gene	Type of mutant	Phenotype	References
Mouse	Grhl1	Targeted null mutant	Delay in coat growth, hair loss, and defective hair anchoring	Wilanowski et al., 2008
Mouse	Grhl3 (Get-1)	Targeted null mutant	NTDs, defective skin barrier and wound repair	Ting et al., 2003a; Yu et al., 2006
Xenopus	Grhl1 (XGrhl1)	Grhl1 targeted antisense morpholino	Defects in head and trunk structures	Tao et al., 2005
Xenopus	Grhl1 (XGrhl1)	Overexpression of dominant-negative Grhl1	Defects in epidermal differentiation	Tao et al., 2005
Xenopus	Grhl3	Overexpression	Influence on cell fate decision during epidermal development	Chalmers et al., 2006
Human	GRHL2 (TFCP2L3)	Familial heterozygous nucleotide substitution; SNP association	Hearing loss, age-related hearing impairment	Peters et al., 2002; van Laer et al., 2008
Drosophila	grainyhead (Elf-1)	Chromosomal deletions identified after screening	Embryonic lethality. Defective epidermal cuticle, grainy head skeletons, and patchy tracheal tubes	Nüsslein-Volhard et al., 1984; Bray and Kafatos, 1991
Drosophila	grainyhead (NTF-1)	Overexpression of grainyhead (NTF-1)	Cuticle and dorsal closure defects	Attardi et al., 1993; Narasimha et al. 2008
Drosophila	grainyhead (NTF-1)	Overexpression of dominant negative grainyhead (NTF-1)	Embryonic lethality	Attardi et al., 1993
Drosophila	grainyhead (grh)	Mutation resulting in absence of functional grainyhead in neuroblasts	Larval-pupal lethality	Uv et al., 1997
C. elegans	Ce-grh-1	RNA interference	Cuticle defect, embryonic lethality	Venkatesan et al., 2003

In Drosophila, grh mutant embryos fail to hatch and have multiple defects, including “grainy” and discontinuous head skeletons, patchy tracheal tubes, and “flimsy” cuticles (Bray and Kafatos, 1991). These defects point to a role for GRH in regulating production and hardening of the cuticle, a protective barrier of cross-linked proteins and chitins that covers the epithelium of the embryonic epidermis (Bray et al., 1989; Stramer and Martin, 2005). The molecular basis for GRH function in the epidermal cells that secrete the cuticle appears to involve transcriptional regulation of the Ddc and pale genes encoding the enzymes DOPA decarboxylase and tyrosine hydroxylase, whose activity is required for cross-linking of cuticle proteins (Uv et al., 1997; Mace et al., 2005). In addition to regulation of initial formation and maintenance of the cuticle, GRH also appears to play a key role in repair of the epidermis following wounding, being required for up-regulation of Ddc in the epidermal cells at the wound margin (Mace et al., 2005).

In addition to being expressed in the cuticle-secreting embryonic epidermis, grh is also expressed in embryonic Drosophila CNS, hence the previous name neuronal transcription factor 1 (Bray et al., 1989; Dynlacht et al., 1989; Attardi et al., 1993). Neuronal cells do not produce cuticle, implying a distinct role of grh in this cell type (although DOPA decarboxylase and tyrosine hydroxylase could still be important targets in neurons). Further diversity of grh function within the larval CNS may be mediated through alternative splicing, which generates two alternative isoforms, GRH-N and GRH-O (Uv et al., 1997). GRH-O appears predominantly in neuroblasts whereas GRH-N is expressed in developing optic lobes, as well as in non-CNS tissues including epidermis, parts of the fore- and hindgut, and the tracheal system (Uv et al., 1997).

Conserved Function of grh Family Genes in Formation and Healing of Epidermal Barriers

In addition to NTDs, null mutant mice for Grhl3 display skin defects affecting barrier function, terminal differentiation, and wound repair (Ting et al., 2005; Yu et al., 2006). These defects correspond with expression of Grhl3 in neonatal and adult skin, where transcripts are detected in the interfollicular epidermis, but are excluded from basal layers (Kudryavtseva et al., 2003; Ting et al., 2003a). The function of Grhl3 in development of the stratum corneum, the protective layer of the epidermis in mammals, therefore, shows remarkable evolutionary conservation with grh function during formation of the analogous cuticular structure in flies (Stramer and Martin, 2005). Moreover, RNA interference-mediated suppression of expression of the C. elegans orthologue of grh, Ce-Grh-1, also results in fragile cuticle in the larva (Venkatesan et al., 2003).

Among Grhl3-regulated proteins, transglutaminase 1, encoded by Tgm1, is thought to play a key role in formation of the epidermal barrier, by cross-linking structural proteins and lipids (Ting et al., 2005). Moreover, the skin barrier function of Tgm1-null mice is impaired, with defects of the stratum corneum, and these mice die neonatally, suggestive of a link between the skin phenotypes of the Tgm1- and Grhl3-deficient mice (Matsuki et al., 1998; Ting et al., 2005). However, analysis of gene expression alterations in skin of Grhl3 knockout mice suggests that regulation of terminal differentiation and barrier function likely involves regulation of multiple genes (Yu et al., 2006). For example, the functional interaction of Grhl3 and Lmo4 may also play a key role (Kudryavtseva et al., 2003; Yu et al., 2006).

Another mammalian grh-like gene, Grhl1, also appears to function in the epidermis, as null mice develop a skin phenotype similar to palmoplantar keratoderma, which is characterized by a thickening of the palms (Wilanowski et al., 2008). These skin defects are thought to result from reduced expression of genes encoding the desmosomal cadherin, desmoglein 1 (Dsg1), because the human and mouse Dsg1 promoters are direct targets of Grhl1 (Wilanowski et al., 2008). Grhl1 mutants also display a delay in coat growth and hair loss as a result of poor anchoring of the hair shaft in the follicles (Wilanowski et al., 2008).

As in mice, a grh family gene is also important for epidermal development in Xenopus. Grhl1 is expressed in the epidermis and has been shown to directly bind a regulatory element in the epidermal keratin gene XK81A1 (Tao et al., 2005). Disruption of Grhl1 activity leads to defects in the epidermis and loss of epidermal keratin expression, which parallels the defects seen in Drosophila grh mutants (Tao et al., 2005).

GRH-LIKE GENES AND HUMAN DISEASE

Human GRHL3 generates three different RNA isoforms, originally designated as SOM1–3, depending on usage of two alternative first coding exons (1A and 1B) and the second exon (Ting et al., 2003b). The SOM2 transcript, which uses exon (1B), is the closest homologue to mouse Grhl3 (Fig. 3), whereas SOM1 and 3 utilize an alternative first exon (Ting et al., 2003b). Alternative splicing also results in omission of exon 2 from the SOM3 variant. Whether the alternative GRHL3 isoforms mediate different biological functions is unclear, but some differences of expression sites are apparent (Ting et al., 2003b). Bioinformatic and experimental analysis of the mouse genomic locus has not revealed an alternative first exon in mouse (see above).

The occurrence of NTDs in curly tail and Grhl3 targeted null mice suggest GRHL3 as a candidate gene for human NTDs, although no definitive link has yet been demonstrated. The GRHL3 gene is located on human chromosome 1p36.11 (www.ensembl.org). Submicroscopic deletions on 1p36 result in a specific syndrome with dysmorphic features and mental retardation (Shapira et al., 1997), although without NTDs. Proximal 1p36 deletions are rare, but patients who are constitutionally hemizygous for the GRHL3 gene have not been identified, so the effect of reduced GRHL3 expression in humans remains unknown (Kang et al., 2007). The genomic region on 1p36 is frequently involved in tumor-specific rearrangements, indicating the presence of loci of general importance for cancer development and progression, although a direct role for GRHL3 in cancer has not been demonstrated to date. Tumors showing high percentages of 1p loss of heterozygosity include oligodendrogliomas, yolk sac tumors, neuroblastomas, and chordomas (Attiyeh et al., 2005; Longoni et al., 2008). As in mice, where diminished Tgm1 expression leads to loss of skin barrier function (Matsuki et al., 1998), mutations in human TGM1 have been identified in families with autosomal recessive lamellar ichthyosis (Russell et al., 1995). It will be interesting to determine whether mutations in GRHL3, an upstream regulator of TGM1 at least in mice, are also found to be associated with disorders of the skin in humans.

A mouse Grhl2 mutant has not yet been reported, but in humans GRHL2 defects are involved with hearing impairment (Table 1). A frameshift mutation in the GRHL2 gene resulting in a premature stop codon has been identified in a large family segregating with an autosomal dominant form of progressive nonsyndromic sensorineural hearing loss (Peters et al., 2002). In addition, a large association study in patients with age-related hearing impairment revealed a significant association with GRHL2 (van Laer et al., 2008).

CONCLUSION

The Grh-like-3 transcription factor is required for neural tube closure, with loss of function resulting in NTDs in targeted null mice (Ting et al., 2003a; Yu et al., 2006). Moreover, reduced expression of Grhl3 appears to be responsible for NTDs in the well-known mouse model curly tail (Ting et al., 2003a; Gustavsson et al., 2007). So far, more than 190 different genes have been found to be required for neural tube closure, based on the occurrence of NTDs in corresponding mutant mice (Copp et al., 2003; Harris and Juriloff, 2007). Despite this, relatively little is known about the genetic risk factors for NTDs in humans. GRHL3 represents a promising candidate for further investigation, as curly tail is thought to be a good model for human NTDs, with its reduced penetrance, phenotypic heterogeneity, and involvement of environmental factors resembling the complex etiology underlying human NTDs. Given the hypothesis that the causative gene in curly tail may be a candidate gene for human NTDs, it will be important to further understand the functional role of Grhl3 during neural tube closure.

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Citing Literature

Volume82, Issue10

Special Issue:Remembering Marcy Speer

October 2008

Pages 728-735

Grainyhead genes and mammalian neural tube closure

Abstract

GRAINYHEAD (GRH) AND THE GRH GENE FAMILY

Grhl3 AND MAMMALIAN NEURAL TUBE CLOSURE

Grhl3 Function Is Required for Spinal Neural Tube Closure

Diminished Grhl3 Expression as the Cause of NTDs in the Curly Tail Mouse

What Is the Nature of the Curly Tail Mutation?

Cranial NTDs Occur with Incomplete Penetrance in Grh-like 3 Mutant Mice

GRH GENES HAVE DIVERSE DEVELOPMENTAL FUNCTIONS

Conserved Function of grh Family Genes in Formation and Healing of Epidermal Barriers

GRH-LIKE GENES AND HUMAN DISEASE

CONCLUSION

REFERENCES

Citing Literature

Figures

References

Information

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Grainyhead genes and mammalian neural tube closure

Abstract

GRAINYHEAD (GRH) AND THE GRH GENE FAMILY

Grhl3 AND MAMMALIAN NEURAL TUBE CLOSURE

Grhl3 Function Is Required for Spinal Neural Tube Closure

Diminished Grhl3 Expression as the Cause of NTDs in the Curly Tail Mouse

What Is the Nature of the Curly Tail Mutation?

Cranial NTDs Occur with Incomplete Penetrance in Grh-like 3 Mutant Mice

GRH GENES HAVE DIVERSE DEVELOPMENTAL FUNCTIONS

Conserved Function of grh Family Genes in Formation and Healing of Epidermal Barriers

GRH-LIKE GENES AND HUMAN DISEASE

CONCLUSION

REFERENCES

Citing Literature

Figures

References

Related

Information