Spalt-related of Drosophila

The spalt-related gene of Drosophila melanogaster is a member of an ancient gene family, defined by the adjacent, region-specific homeotic gene spalt. Both genes have three widely spaced sets of C2H2 zinc finger motifs, but spalt-related has a fourth pair of C-terminal fingers resembling the Xenopus homolog, Xsal-1. The spalt-related gene is not expressed in early development as is spalt, but is expressed from mid-embryogenesis to the adult stage, but not in ovaries. Expression is localized to the nervous system. spalt-related is more promenant that spalt in the embryonic brain, but spalt is expressed at a higher level than spalt-related in the posterior spiracles. There is some degree of differential expression in the ventral cord of the CNS (Barrio, 1996).

Spalt homologs in C. elegans

Neuronal and mesodermal cell types are generated in separate cell lineages during the larval development of Caenorhabditis elegans. The gene sem-4 is required in both types of lineages for the normal development of neuronal and mesodermal cell types. The sem-4 gene encodes a protein containing seven zinc finger motifs of the C2H2 class, four of which are arranged in two pairs widely separated in the primary sequence of the protein. These pairs of zinc fingers are similar to pairs of zinc fingers in the protein encoded by the Drosophila homeotic gene spalt and in the human transcription factor PRDII-BF1. Analysis of sem-4 alleles suggests that different zinc fingers in the SEM-4 protein may function differentially in neuronal and mesodermal cell types. It is proposed that sem-4 interacts with different transcription factors in different cell types to control the transcription of genes that function in the processes of neuronal and mesodermal cell development (Basson, 1996).

Vulval cell-fate determination in C. elegans requires the action of numerous gene products, including components of the Ras/Raf/MAPK signaling cascade and the hox gene lin-39, an Antennapedia class homeodomain, most similar to those of the Drosophila homeotic genes Deformed and Sex combs reduced. sem-4 encodes a zinc finger protein, homologous to Drosophila Spalt, with roles in the fate specification of sex myoblasts, coelomocytes, and multiple neuronal lineages in C. elegans. By characterizing three new alleles of sem-4, identified in a screen for vulval-defective mutants, it has been determined that loss of sem-4 activity results in abnormal specification of the secondary vulval cell lineages. sem-4 interactions with other genes involved in vulval differentiation were analyzed and it was determined that sem-4 does not function directly in the Ras-mediated signal transduction pathway but acts in close association with and upstream of lin-39 to promote vulval cell fate. sem-4 regulates lin-39 expression and it is proposed that sem-4 is a regulator of lin-39 in the vulval cell-fate determination pathway that may act to link lin-39 to incoming signals (Grant, 2000).

Members of the spalt (sal) gene family encode zinc-finger proteins that are putative tumor suppressors and regulate anteroposterior (AP) patterning, cellular identity, and, possibly, cell cycle progression. The mechanism through which sal genes carry out these functions is unclear. The Caenorhabditis elegans sal gene sem-4 controls the fate of several different cell types, including neurons, muscle and hypodermis. Mutation of sem-4 transforms particular tail neurons into touch-neuron-like cells. In wild-type C. elegans, six touch receptor neurons mediate the response of the worm to gentle touch. All six touch neurons normally express the LIM homeobox gene mec-3 (Drosophila homolog: Lim3). A subset, the two PLM cells, also express the Hox gene egl-5, an Abdominal-B homolog, which is required for correct mec-3 expression in these cells. The abnormal touch-neuron-like-cells in sem-4 animals express mec-3; a subset also express egl-5. The following observations are reported: (1) that ectopic expression of sem-4 in normal touch cells represses mec-3 expression and reduces touch cell function; (2) that egl-5 expression is required for both the fate of normal PLM touch neurons in wild-type animals and the fate of a subset of abnormal touch neurons in sem-4 animals, and (3) that SEM-4 specifically binds a shared motif in the mec-3 and egl-5 promoters that mediates repression of these genes in cells in the tail. It is concluded that sem-4 represses egl-5 and mec-3 through direct interaction with regulatory sequences in the promoters of these genes; that sem-4 indirectly modulates mec-3 expression through its repression of egl-5, and that this negative regulation is required for proper determination of neuronal fates. It is suggested that the mechanism and targets of regulation by sem-4 are conserved throughout the sal gene family: other sal genes might regulate patterning and cellular identity through direct repression of Hox selector genes and effector genes (Toker, 2003).

Hox genes appear to be targets not only of sem-4 but also of other sal genes. Drosophila sal might negatively regulate Sex combs reduced (Scr) and other Drosophila Hox genes. Loss of sal function in Drosophila BX-C minus embryos produces some limited ectopic expression of the Hox gene Scr. Mutations in sal enhance the phenotypes of Polycomb group (PcG) mutants. These genes are known to be negative regulators of Hox genes. Loss of sal function affects AP patterning in Drosophila. Mutations in sal incompletely transform both head and tail structures into trunk-like structures: sal activity has been shown to promote head development. Hox genes in mammals might also be targets of sal family genes. Patients with TBS, which is caused by mutations in SALL1, display characteristic features of syndromes associated with mutations in HOX genes (Toker, 2003).

LIM homeobox genes, such as mec-3, might also be conserved targets of sal genes. The closest mammalian homolog to mec-3 is the human LIM homeobox gene Lhx5. Lhx5 and the human SALL1 gene appear to be expressed in different sets of cells in the developing thalamus, which constitutes a very small portion of the entire brain. SALL1 and Lhx5 are not expressed in most other regions of the fetal brain. Their expression in separate thalamic cells could indicate that SALL1 restricts Lhx5 expression in the thalamus (Toker, 2003).

The mechanism through which sem-4 negatively regulates its targets is probably conserved. SEM-4, SALL1 and mouse sall1 are transcriptional repressors. SALL1 and mouse sall1, fused to heterologous DNA binding domains, behave as repressors in mammalian cell culture assays. It is suggested that these genes bind directly to regulatory regions of their targets (Toker, 2003).

Drosophila and mammalian studies have suggested that sal genes might function as PcG genes. Mutations of Drosophila sal cause limited ectopic expression of the Hox genes Ubx and Scr, and sal mutations enhance mutations in the PcG genes polyhomeotic and Polycomb-like. Human SALL1 localizes to chromocenters in mammalian cells and mouse sall1 interacts with components of chromatin remodeling complexes. One additional speculation is that Drosophila sal might bind to a 138 bp silencing sequence in the Polycomb response element in Abd-B, the egl-5 ortholog. Two sites have been identified that match the SEM-4 binding sequence in this Drosophila silencing element (Toker, 2003).

A regulatory program for excretory system regeneration in planarians

Planarians can regenerate any missing body part, requiring mechanisms for the production of organ systems in the adult, including their prominent tubule-based filtration excretory system called protonephridia. This study identified a set of genes, Six1/2-2, POU2/3, hunchback, Eya and Sall, that encode transcription regulatory proteins that are required for planarian protonephridia regeneration. During regeneration, planarian stem cells are induced to form a cell population in regeneration blastemas expressing Six1/2-2, POU2/3, Eya, Sall and Osr that is required for excretory system formation. POU2/3 and Six1/2-2 are essential for these precursor cells to form. Eya, Six1/2-2, Sall, Osr and POU2/3-related genes are required for vertebrate kidney development. Planarian and vertebrate excretory cells express homologous proteins involved in reabsorption and waste modification. Furthermore, novel nephridia genes were identified. These results identify a transcriptional program and cellular mechanisms for the regeneration of an excretory organ and suggest that metazoan excretory systems are regulated by genetic programs that share a common evolutionary origin (Scimone, 2011).

The generation and diversification of butterfly eyespot color patterns involve Spalt

A fundamental challenge of evolutionary and developmental biology is understanding how new characters arise and change. The recently derived eyespots on butterfly wings vary extensively in number and pattern between species and play important roles in predator avoidance. Eyespots form through the activity of inductive organizers (foci) at the center of developing eyespot fields. Foci are the proposed source of a morphogen, the levels of which determine the color of surrounding wing scale cells. However, it is unknown how reception of the focal signal translates into rings of different-colored scales, nor how different color schemes arise in different species. Several transcription factors, including butterfly homologs of the Drosophila Engrailed/Invected and Spalt proteins have been identified. These are deployed in concentric territories corresponding to the future rings of pigmented scales that compose the adult eyespot. A new Bicyclus anynana wing pattern mutant, Goldeneye, has been isolated in which the scales of one inner color ring become the color of a different ring. These changes correlate with shifts in transcription factor expression, suggesting that Goldeneye affects an early regulatory step in eyespot color patterning. In different butterfly species, the same transcription factors are expressed in eyespot fields, but in different relative spatial domains that correlate with divergent eyespot color schemes. These results suggest that signaling from the focus induces nested rings of regulatory gene expression that subsequently control the final color pattern. Furthermore, the remarkably plastic regulatory interactions downstream of focal signaling have facilitated the evolution of eyespot diversity (Brunetti, 2001).

To distinguish between different potential mechanisms of eyespot development and evolution, candidate genes involved in eyespot color pattern formation were sought. A screen was performed for gene products that are expressed during the period of scale cell differentiation (12 to 36 hours after pupation) and that have patterns that are correlated with the concentric rings of Bicyclus anynana eyespots. Among the various proteins and transcripts surveyed (these included Cubitus interruptus, Schnurri, SMAD, Brinker, aristaless, dachshund, and teashirt), only the Engrailed/Invected (Engrailed and/or Invected, hereafter denoted by En/Inv) and Spalt (Sal) transcription factors are expressed in patterns of scale-forming cells that correlate with eyespot formation. All identified proteins are expressed in cells in the region of the focus at the center of each eyespot field. Remarkably, a second domain of En/Inv expression arises in the 16 hour pupal wing in a distinct ring of cells outside of the focal region and at the periphery of each eyespot field. In addition, Sal is expressed in rings of cells between the focal region and the ring of En/Inv-expressing cells. Based upon physical landmarks of the developing wing and by comparison of the relative size and position of the concentric rings of gene expression patterns with the colored rings of the adult eyespot, correlations between protein expression patterns and the three colored rings of B. anynana eyespots were found. The En/Inv, Sal, and Dll expression in the focus corresponds to the white center in the adult eyespot. The territory marked by Sal and Dll expression, but not En/Inv expression, appears to correspond to the domain of the black ring of scales in the adult eyespot. Additionally, the outer ring of En/Inv expression correlates with the position of the gold ring of scales in the adult wing. A gene product for which the pattern of expression correlates with the outermost dark-brown ring of scales has not been identified (Brunetti, 2001).

From observations of the temporal and spatial relationships between En/Inv, Sal, and Dll expression, two important inferences can be made: (1) the switch from synchronous coincident expression of these three proteins in the center of the eyespot field to their asynchronous, nonoverlapping expression in the outer rings of the field suggests that they are under different regulatory controls when the foci are first established than when the eyespot field is elaborated; (2) the sequential appearance of the rings, in particular the expression of En/Inv in cells just outside of the Sal domain, suggests that one mechanism for generating concentric patterns of gene expression may be to exclude the expression of one gene from another's domain (Brunetti, 2001).

The transplantation of eyespot foci between species or of selected lines of B. anynana differing in eyespot color composition induces eyespot patterns characteristic of the host animal, suggesting that the response to the focal signal (not the signal itself) is different between species. It is possible that the differences in cells' responses to focal signaling could arise as a result of changes in the expression patterns of regulators. Alternatively, direct responses to focal signaling may be similar between species, but the regulators may interact with different downstream genes involved in scale pigmentation and structure. To determine when during development differences arise between the eyespot color schemes of various species, the expression patterns were compared of En/Inv, Sal, and Dll in B. anynana (Nymphalidae, Satyrinae), Precis coenia (Nymphalidae, Nymphalinae), Vanessa cardui (Nymphalidae, Nymphalinae), and Lycaeides melissa (Lycaenidae, Lycaeninae). In each of the examined species, which represent two different families of butterflies and three different genera within the Nymphalidae, the expression patterns of En/Inv, Sal, and Dll are different, yet they mark territories in the pupal wing that often correlate with color pattern schemes on the adult wing. For example, in P. coenia, the Sal territory in the pupal wing marks the entire area encompassed by the adult eyespot. In addition, the coexpression of En/Inv, Sal, and Dll in P. coenia forewings in an asymmetric patch of scales at the center of the pupal eyespot corresponds to the white/blue scales at the center of the adult eyespot. The coexpression of the same genes in scale-building cells outside of this central spot correlates with the black ring of scales on the adult. In V. cardui, a species closely related to P. coenia, En/Inv is expressed in an outer ring of scale-building cells that correlates with the black ring of scales in the adult eyespot. However, in L. melissa, a crescent of En/Inv expression correlates with the future position of orange scales on the adult, and En/Inv and Sal coexpression correlates with the metallic-looking patch of scales at the center of the eyespot field (Brunetti, 2001).

From comparative data, it is concluded that eyespot color pattern diversity is generated by regulatory differences at two distinct stages of eyespot development that evolve independently of each other: (1) during the focal signaling stage, through the generation of different combinations and patterns of expression of regulatory genes such as en/inv, sal, and Dll; and (2) during the scale differentiation stage, through differences in the response of pigmentation genes to the upstream regulators (Brunetti, 2001).

These results indicate that at least one tier of spatially regulated transcription factors is interposed between focal signaling and scale color differentiation. How the graded distribution of a focal signal is translated into the concentric territories of En/Inv, Sal, and Dll expression is therefore of special interest. In B. anynana, it is suggested that this occurs through response thresholds of, and negative cross-regulation among, genes regulated by the signal. For example, one of the simplest explanations for the exclusion of En/Inv and Sal expression from each other's territories outside of the focus could be the repression of one gene by the product of the other. The reciprocal effects of the Goldeneye mutation on En/Inv and Sal expression are strongly suggestive of negative crossregulation. The establishment, through negative crossregulation, of distinct spatial domains of downstream genes in response to a single activator is a common theme illustrated by the subdivision of the Drosophila embryonic mesoderm and neuroectoderm and of the proximodistal axis of Drosophila limb fields. In P. coenia, however, the nested nonexclusive expression of Sal and En/Inv suggests that here these genes do not crossregulate. Rather, the nested expression pattern outside of the focus is most simply explained by different threshold responses of these two genes to the focal signal; these responses are analogous to the threshold responses of genes to long-range signals in the Xenopus mesoderm and the Drosophila imaginal wing field (Brunetti, 2001).

The deployment of En/Inv, Sal, and Dll in all of the species examined also raises some interesting possible scenarios regarding the origin and diversification of eyespots and the evolution of the underlying genetic regulatory system that controls eyespot pattern formation. It has been proposed that eyespots have a single origin and are derived from simpler spot patterns of uniform color that evolved into organizing centers. Because all three proteins are deployed in color-correlated patterns in this well-diverged group of butterflies, it is likely that these genes were recruited into the developmental program early during the evolution of eyespots. Furthermore, it is intriguing that while the three proteins have distinct expression patterns during scale differentiation, they are coexpressed during focus formation. It is tempting to speculate, on the basis of the data presented here, that the evolution of eyespots in response to diverse selective environments involved the modification of the deployment of genes that were originally expressed in simpler spot patterns into additional concentric patterns organized around and by cells in the center of the eyespot field (Brunetti, 2001).

Spalt function in fish and frogs

The medaka fish (a Japanese freshwater poeciliid fish) gene spalt encodes a zinc-finger transcription factor, which is expressed in all known hedgehog signaling centers of the embryo and in the organizer region at the midbrain-hindbrain boundary. The spalt expression domains expand in response to ectopic hedgehog activity and narrow in the presence of protein kinase A activity, an antagonist of hedgehog signaling, indicating that spalt is a hedgehog target gene. These results also suggest a signaling mechanism for anterior-posterior patterning of the vertebrate brain that controls spalt expression at the midbrain-hindbrain boundary in a protein kinase A dependent manner, likely to involve an unknown member of the hedgehog family (Koster, 1997).

Xsal-1 is a Xenopus homolog of spalt. The frog protein has three double zinc fingers like SAL, but in addition, it has a fourth double finger and an additional single finger. Xsal-1 is expressed in lateral axon tracts, in the midbrain, hindbrain and limbs. The frog gene is regulated by signals from the notochord and floor plate, and might function in neuronal cell specification (Hollenmann, 1996).

The spalt gene family is characterized by unique double zinc finger motifs and is conserved among various species from Drosophila to humans. A new Xenopus member of this family, Xsal-3, has been identified. It is 38% homologous at the amino acid level to the previously reported Xenopus homolog of the spalt gene, Xsal-1. Alternatively spliced Xsal-3 transcripts give rise to RNAs coding either two or three double zinc fingers, and the longer form is expressed maternally. Xsal-3 is expressed in the neural tube, the mandibular, hyoid, and branchial arch, and the pronephric duct, which is different from the expression pattern of Xsal-1. These findings suggest that Xsal-3 may have distinct roles in early Xenopus development (Onuma, 1999).

XsalF, a frog homolog of the Drosophila homeotic selector Spalt, plays an essential role for the forebrain/midbrain determination in Xenopus. XsalF overexpression expands the domain of forebrain/midbrain genes and suppresses midbrain/hindbrain boundary (MHB) markers and anterior hindbrain genes. Loss-of-function studies show that XsalF is essential for the expression of the forebrain/midbrain genes and for the repression of the caudal genes. Interestingly, XsalF functions by antagonizing canonical Wnt signaling, which promotes caudalization of neural tissues. XsalF is required for anterior-specific expressions of GSK3ß and Tcf3, genes encoding antagonistic effectors of Wnt signaling. Loss-of-function phenotypes of GSK3ß and Tcf3 mimic those of XsalF while injections of GSK3ß and Tcf3 rescue loss-of-function phenotypes of XsalF. These findings suggest that the forebrain/midbrain-specific gene XsalF negatively controls cellular responsiveness to posteriorizing Wnt signals by regulating region-specific GSK3ß and Tcf3 expression (Onai, 2004).

Spalt-like 4 promotes posterior neural fates via repression of pou5f3 family members in Xenopus

Amphibian neural development occurs as a two-step process: (1) induction specifies a neural fate in undifferentiated ectoderm; and (2) transformation induces posterior spinal cord and hindbrain. Signaling through the Fgf, retinoic acid (RA) and Wnt/beta-catenin pathways is necessary and sufficient to induce posterior fates in the neural plate, yet a mechanistic understanding of the process is lacking. This study screened for factors enriched in posterior neural tissue and identified spalt-like 4 (sall4), which is induced by Fgf. Knockdown of Sall4 results in loss of spinal cord marker expression and increased expression of pou5f3.2 (oct25), pou5f3.3 (oct60) and pou5f3.1 (oct91) (collectively, pou5f3 genes), the closest Xenopus homologs of mammalian stem cell factor Pou5f1 (Oct4). Overexpression of the pou5f3 genes results in the loss of spinal cord identity and knockdown of pou5f3 function restores spinal cord marker expression in Sall4 morphants. Finally, knockdown of Sall4 blocks the posteriorizing effects of Fgf and RA signaling in the neurectoderm. These results suggest that Sall4, activated by posteriorizing signals, represses the pou5f3 genes to provide a permissive environment allowing for additional Wnt/Fgf/RA signals to posteriorize the neural plate (Young, 2014).

Spalt function in mammals

Msal is a mouse homolog that contains alternatively seven and nine zinc fingers, each of which contains tha SAL box motif KTTKGNLK. The additional zinc finger pair is conserved in mouse and Xenopus and expressed as an alternatively spliced product. A glutamine-rich putative transactivation domain close to the amino-terminus is conserved in the mouse homolog. msal is expressed in the developing neuroectoderm of the brain, the inner ear and the spinal cord and in urogenital ridge-derived structures. A weaker and transient expression is seen in early embryos in the branchial arches and in notochord, limb buds and heart (Ott, 1996).

Mutations of SALL1 related to spalt of Drosophila have been found to cause Townes-Brocks syndrome, suggesting a function of SALL1 for the development of anus, limbs, ears, and kidneys. No function is yet known for SALL2, another human spalt-like gene. The structure of SALL2 is different from SALL1 and all other vertebrate spalt-like genes described in mouse, Xenopus, and Medaka, suggesting that SALL2-like genes might also exist in other vertebrates. Consistent with this hypothesis, a SALL2 homologous mouse gene, Msal-2, has been isolated and characterized. In contrast to other vertebrate spalt-like genes both SALL2 and Msal-2 encode only three double zinc finger domains, the most carboxyterminal of which only distantly resembles spalt-like zinc fingers. The evolutionary conservation of SALL2/Msal-2 suggests that two lines of sal-like genes with presumably different functions arose from an early evolutionary duplication of a common ancestor gene. Msal-2 is expressed throughout embryonic development but also in adult tissues, predominantly in brain. The function of SALL2/Msal-2 still needs to be determined (Kohlhase, 2000a).

Two human sal-like genes have been isolated to date: SALL1 on chromosome 16q12.1 and SALL2 on chromosome 14q11.1-q12.1. Truncating mutations of SALL1 have been shown to cause Townes-Brocks syndrome and are thought to result in SALL1 haploinsufficiency. Sequence comparison of SALL1 to the related genes Msal in mouse and Xsal-1 in Xenopus suggest that SALL1 is not the human orthologue of Msal and Xsal-1. By database searching and genomic cloning, an EST and a corresponding human cosmid clone have been isolated that contain the coding sequence of a human gene highly similar to mouse Msal. This gene, named SALL3, is expressed in different regions of human fetal brain and in different adult human tissues. The chromosomal localization of SALL3 at 18q23 suggests that haploinsufficiency of this gene might contribute to the phenotype of patients with 18q deletion syndrome (Kohlhause, 2000b).

While some of the signaling molecules that govern establishment of the limb axis have been characterized, little is known about the downstream effector genes that interpret these signals. In Drosophila, the spalt gene is involved in cell fate determination and pattern formation in different tissues. A chick homolog of Drosophila spalt, csal1, has been cloned. csal1 is expressed in limb buds from HH stages 17 to 26, in both the apical ectodermal ridge and the distal mesenchyme. Signals from the apical ridge are essential for csal1 expression, while the dorsal ectoderm is required for csal1 expression at a distance from the ridge. These data indicate that both FGF and Wnt signals are required for the regulation of csal1 expression in the limb. Mutations in the human homolog of csal1, termed Hsal1/SALL1, result in a condition known as Townes-Brocks syndrome (TBS), which is characterized by preaxial polydactyly. The developmental expression of csal1 together with the digit phenotype in TBS patients suggests that csal1 may play a role in some aspects of distal patterning (Farrell, 2000).

Townes-Brocks syndrome (TBS) is a rare autosomal-dominant malformation syndrome with a combination of anal, renal, limb and ear anomalies. Cytogenetic findings suggest that the gene mutated in TBS maps to chromosome 16q12.1, where SALL1 (previously known as HSAL1), a human homolog of Drosophila spalt (sal), is located. No phenotype has yet been attributed to mutations in vertebrate sal-like genes. The expression patterns of sal-like genes in mouse, Xenopus and the fish Medaka, and the finding that Medaka sal is regulated by Sonic hedgehog, prompted an examination of SALL1 as a TBS candidate gene. SALL1 mutations have been shown to cause TBS in a family with vertical transmission of TBS and in an unrelated family with a sporadic case of TBS. Both mutations are predicted to result in a prematurely terminated SALL1 protein lacking all putative DNA binding domains. TBS therefore represents another human developmental disorder caused by mutations in a putative C2H2 zinc-finger transcription factor (Kohlhase, 1998).

Townes-Brocks syndrome (TBS) is an autosomal dominant developmental disorder characterized by anal and thumb malformations and by ear anomalies that can affect the three compartments and usually lead to hearing loss. The gene underlying TBS, SALL1, is a human homolog of the Drosophila spalt gene, which encodes a transcription factor. A search for SALL1 mutations undertaken in 11 unrelated affected individuals (five familial and six sporadic cases) led to the detection of mutations in nine of them. One nonsense and six different novel frameshift mutations, all located in the second exon, were identified. These mutations establish that TBS results from haploinsufficiency. The finding of de novo mutations in the sporadic cases is consistent with the proposed complete penetrance of the disease. Moreover, the occurrence of the same 826 C-to-T transition in a CG dimer, in six sporadic cases (i.e., six of the eight mutations identified in sporadic cases), reveals the existence of a mutation hotspot. Six different SALL1 polymorphisms were identified in the course of the present study, three of which are clustered in a particular region of the gene that encodes a stretch of serine residues. Finally, the chromosome 16 breakpoint of a t(5;16)(p15.3;q12.1) translocation carried by a TBS-affected individual was mapped at least 180 kb telomeric to SALL1, thus indicating that a position effect underlies the disease in this individual (Kohlhase, 1999).

Townes-Brocks syndrome (TBS) is an autosomal dominant developmental disorder characterized by anal and thumb malformations and by ear anomalies that can affect the three compartments and usually lead to hearing loss. A search for SALL1 mutations undertaken in 11 unrelated affected individuals (five familial and six sporadic cases) led to the detection of mutations in nine of them. One nonsense and six different novel frameshift mutations, all located in the second exon, have been identified. Together with the previously reported mutations, they establish that TBS results from haploinsufficiency. The finding of de novo mutations in the sporadic cases is consistent with the proposed complete penetrance of the disease. Moreover, the occurrence of the same 826C to T transition in a CG dimer, in three sporadic cases from the present series and three sporadic cases from the other series (i.e., six of the eight mutations identified in sporadic cases), reveals the existence of a mutation hotspot. Six different SALL1 polymorphisms have been identified in the course of the present study, three of which are clustered in a particular region of the gene that encodes a stretch of serine residues. Finally, the chromosome 16 breakpoint of a t(5;16)(p15.3;q12.1) translocation carried by a TBS-affected individual was mapped at least 180 kb telomeric to SALL1, thus indicating that a position effect underlies the disease in this individual (Marlin, 1999).

spalt of Drosophila melanogaster is an important developmental regulator gene and encodes a zinc finger protein of unusual but characteristic structure. Two human sal-like genes have been isolated so far: SALL1 on chromosome 16q12.1 and SALL2 on chromosome 14q11.1-q12.1. Truncating mutations of SALL1 have been shown to cause Townes-Brocks syndrome and are thought to result in SALL1 haploinsufficiency. Sequence comparison of SALL1 to the related genes Msal in mouse and Xsal-1 in Xenopus laevis suggests that SALL1 is not the human ortholog of Msal and Xsal-1. By database searching and genomic cloning, an EST and a corresponding human cosmid clone, have been isolated that contain coding sequence of a human gene highly similar to mouse Msal. This gene, named SALL3, was found to be expressed in different regions of human fetal brain and in different adult human tissues. The chromosomal localization of SALL3 at 18q23 suggests that haploinsufficiency of this gene might contribute to the phenotype of patients with 18q deletion syndrome (Chen, 1999).

SALL1 is a mammalian homolog of the Drosophila region-specific homeotic gene spalt (sal); heterozygous mutations in SALL1 in humans lead to Townes-Brocks syndrome. A mouse homolog of SALL1 (Sall1) has been isolated and it has been found that mice deficient in Sall1 die in the perinatal period and that kidney agenesis or severe dysgenesis are present. Sall1 is expressed in the metanephric mesenchyme surrounding ureteric bud; homozygous deletion of Sall1 results in an incomplete ureteric bud outgrowth, a failure of tubule formation in the mesenchyme and an apoptosis of the mesenchyme. This phenotype is likely to be primarily caused by the absence of the inductive signal from the ureter, as the Sall1-deficient mesenchyme is competent with respect to epithelial differentiation. Sall1 is therefore essential for ureteric bud invasion, the initial key step for metanephros development (Nishinakamura, 2001).

Drosophila spalt is downstream of the wingless signal in the tracheal system and sal deletion results in the absence of dorsal trunks in the trachea. Though murine Wnt4 is essential for kidney development, a normal ureter-mesenchyme interaction occurs in its mutants, which are different from phenotypes of Sall1-deficient mice. Furthermore Sall1 is not expressed in trachea and lungs, and Sall1-deficient mice apparently have no lung defects. Therefore, the simple analogy of Drosophila does not apply to mammals (Nishinakamura, 2001).

SALL4, a human homolog to Drosophila spalt, is a novel zinc finger transcriptional factor essential for development. SALL4 and its isoforms (SALL4A and SALL4B) were cloned. Through immunohistochemistry and RT-PCR, it was demonstrated that SALL4 was constitutively expressed in human primary acute myeloid leukemia (AML), and the leukemogenic potential of constitutive expression of SALL4 was directly tested in a murine model. SALL4B transgenic mice developed myelodysplastic syndrome (MDS)-like features and subsequently AML that was transplantable. Increased apoptosis associated with dysmyelopoiesis was evident in transgenic mouse marrow and colony-formation (CFU) assays. Both isoforms could bind to beta-catenin and synergistically enhanced the Wnt/beta-catenin signaling pathway. These data suggest that the constitutive expression of SALL4 causes MDS/AML, most likely through the Wnt/beta-catenin pathway. The murine model provides a useful platform to study human MDS/AML transformation, as well as the Wnt/beta-catenin pathway's role in the pathogenesis of leukemia stem cells (Ma, 2006).

Mutations in SALL4, the human homolog of the Drosophila homeotic gene spalt (sal), cause the autosomal dominant disorder known as Okihiro syndrome. This study shows that a targeted null mutation in the mouse Sall4 gene leads to lethality during peri-implantation. Growth of the inner cell mass from the knockout blastocysts is reduced, and Sall4-null embryonic stem (ES) cells proliferat poorly with no aberrant differentiation. Furthermore, anorectal and heart anomalies in Okihiro syndrome are caused by Sall4 haploinsufficiency and that Sall4/Sall1 heterozygotes exhibited an increased incidence of anorectal and heart anomalies, exencephaly and kidney agenesis. Sall4 and Sall1 formed heterodimers, and a truncated Sall1 caused mislocalization of Sall4 in the heterochromatin; thus, some symptoms of Townes-Brocks syndrome caused by SALL1 truncations could result from SALL4 inhibition (Sakaki-Yumoto, 2006).

Vertebrate placodes are regions of thickened head ectoderm that contribute to paired sensory organs and cranial ganglia. The transcription factor Spalt4 (also known as Sall4) is broadly expressed in chick preplacodal epiblast and later resolves to otic, lens and olfactory placodes. Ectopic expression of Spalt4 by electroporation is sufficient to induce invagination of non-placodal head ectoderm and prevent neurogenic placodes from contributing to cranial ganglia. Conversely, loss of Spalt4 function in the otic placode results in abnormal otic vesicle development. Intriguingly, Spalt4 appears to initiate a placode program appropriate for the axial level but is not involved in later development of specific placode fates. Fgfs can regulate Spalt4, since implantation of Fgf2 beads into the area opaca induces its expression. The results suggest that Spalt4 is involved in early stages of placode development, initiating cranial ectodermal invagination and region-specific gene regulatory networks (Barembaum, 2007).

Sall genes regulate region-specific morphogenesis in the mouse limb by modulating Hox activities

The genetic mechanisms that regulate the complex morphogenesis of generating cartilage elements in correct positions with precise shapes during organogenesis, fundamental issues in developmental biology, are still not well understood. By focusing on the developing mouse limb, importance was confirmed of transcription factors encoded by the Sall gene family in proper limb morphogenesis, and it was further shown that they have overlapping activities in regulating regional morphogenesis in the autopod (the distal elements of a limb that will give rise to the wrist and the fingers in the forelimb, and the ankle and toes in the hindlimb). Sall1/Sall3 double null mutants exhibit a loss of digit1 as well as a loss or fusion of digit2 and digit3, metacarpals and carpals in the autopod. Sall activity affects different pathways, including the Shh signaling pathway, as well as the Hox network. Shh signaling in the mesenchyme is partially impaired in the Sall mutant limbs. Additionally, the data suggest an antagonism between Sall1-Sall3 and Hoxa13-Hoxd13. Expression of Epha3 and Epha4 is downregulated in the Sall1/Sall3 double null mutants, and, conversely, is upregulated in Hoxa13 and Hoxd13 mutants. Moreover, the expression of Sall1 and Sall3 is upregulated in Hoxa13 and Hoxd13 mutants. Furthermore, by using DNA-binding assays, it was shown that Sall and Hox compete for a target sequence in the Epha4 upstream region. In conjunction with the Shh pathway, the antagonistic interaction between Hoxa13-Hoxd13 and Sall1-Sall3 in the developing limb may contribute to the fine-tuning of local Hox activity that leads to proper morphogenesis of each cartilage element of the vertebrate autopod (Kawakami, 2009).

Sall1 balances self-renewal and differentiation of renal progenitor cells

The formation of the proper number of functional nephrons requires a delicate balance between renal progenitor cell self-renewal and differentiation. The molecular factors that regulate the dramatic expansion of the progenitor cell pool and differentiation of these cells into nephron precursor structures (renal vesicles) are not well understood. This study shows that Sall1, a nuclear transcription factor, is required to maintain the stemness of nephron progenitor cells. Transcriptional profiling of Sall1 mutant cells revealed a striking pattern, marked by the reduction of progenitor genes and amplified expression of renal vesicle differentiation genes. These global changes in gene expression were accompanied by ectopic differentiation at E12.5 and depletion of Six2+Cited1+ cap mesenchyme progenitor cells. These findings highlight a novel role for Sall1 in maintaining the stemness of the progenitor cell pool by restraining their differentiation into renal vesicles (Basta, 2014).

The Spalt family transcription factor Sall3 regulates the development of cone photoreceptors and retinal horizontal interneurons

The mammalian retina is a tractable model system for analyzing transcriptional networks that guide neural development. Spalt family zinc-finger transcription factors play a crucial role in photoreceptor specification in Drosophila, but their role in mammalian retinal development has not been investigated. This study shows that that the spalt homolog Sall3 is prominently expressed in developing cone photoreceptors and horizontal interneurons of the mouse retina and in a subset of cone bipolar cells. Sall3 is both necessary and sufficient to activate the expression of multiple cone-specific genes, and Sall3 protein is selectively bound to the promoter regions of these genes. Notably, Sall3 shows more prominent expression in short wavelength-sensitive cones than in medium wavelength-sensitive cones, and Sall3 selectively activates expression of the short but not the medium wavelength-sensitive cone opsin gene. It was further observed that Sall3 regulates the differentiation of horizontal interneurons, which form direct synaptic contacts with cone photoreceptors. Loss of function of Sall3 eliminates expression of the horizontal cell-specific transcription factor Lhx1, resulting in a radial displacement of horizontal cells that partially phenocopies the loss of function of Lhx1. These findings not only demonstrate that Spalt family transcription factors play a conserved role in regulating photoreceptor development in insects and mammals, but also identify Sall3 as a factor that regulates terminal differentiation of both cone photoreceptors and their postsynaptic partners (de Melo, 2011).

These findings demonstrate that Sall3 plays a crucial role in regulating the development of both cone photoreceptors and horizontal interneurons (see Role of Sall3 in horizontal cell and S-cone photoreceptor development). Sall3 is necessary for the expression of a range of cone-specific genes, with only a very small number of short wavelength-sensitive cone opsin (Sop) positive and cone Arrestin (Arr3) positive cells detectable in Sall3-/- retinas. Microarray experiments revealed that many other cone-enriched transcripts are selectively downregulated in Sall3-/- retinas, including Pde6c, Pde6h and Crb1, whereas some, such as Gnat2 and Gnb3, show little or no change. Strikingly, overexpression of Sall3 by electroporation was sufficient to induce the expression of both Sop and Arr3, not only in photoreceptors but also in a subset of cells in the inner nuclear layer (INL), without affecting expression of medium wavelength-sensitive cone opsin (Mop). Rod photoreceptors overexpressing Sall3 in electroporated retinas were morphologically indistinguishable from those of controls. These rod photoreceptors that overexpressed Sall3 retained their normal morphology, laminar position and pattern of expression of cell-specific markers (de Melo, 2011).

These data imply that Sall3 is activated in Sop-expressing photoreceptors undergoing terminal differentiation, and that Sall3 itself might directly activate a subset of cone-specific genes in a coordinated manner, analogous to the orphan nuclear hormone receptor Errβ (Esrrb) in rod photoreceptors. Several different nuclear hormone receptors, along with the transcriptional co-regulator Pias3, have been shown to be required for medium wavelength-sensitive cones to activate the expression of Mop while simultaneously repressing the expression of Sop. Sall3, however, represents the first cone-expressed transcription factor that selectively actives the expression of Sop (de Melo, 2011).

The strong activation of S-cone-specific transcripts by Sall3 implies a surprising functional homology with the Spalt gene complex of Drosophila in the regulation of photoreceptor differentiation. In Drosophila, Spalt genes are necessary for specification of the inner R7 and R8 photoreceptors, which are responsible for color discrimination and in many respects form a cone-like photoreceptor class in the compound eye. Strikingly, loss of function of Spalt genes results in the ectopic expression of Rh1 opsin (NinaE), which is normally expressed in the outer R1-R6 photoreceptors, in the R7 and R8 cells, whereas expression of Rh3-Rh6 is lost. However, axonal projections of inner photoreceptors are unaltered in Drosophila sal mutants. This partial shift in photoreceptor identity resembles that seen following Sall3 overexpression in rod photoreceptors. These cells appear morphologically normal and continue to express rhodopsin, but now robustly express cone-specific genes. Notably, Spalt genes are selectively expressed in blue-sensitive Rh5-positive R8 photoreceptors and are required for expression of Rh5 opsin (de Melo, 2011).

In mice, it was observed that Sall3 is both necessary and sufficient for the expression of blue-sensitive cone opsin but not green-sensitive cone opsin. Such a direct conservation of gene function in photoreceptor development is unusual, and even more surprising because the blue-sensitive visual opsins of insects and vertebrates evolved independently (Shichida, 2009). Although this observation might represent evolutionary convergence, it could alternatively imply that ancestral bilateria possessed a dedicated short wavelength-sensitive photoreceptor cell type, the differentiation of which was guided by a Spalt family gene, with the blue-sensitive opsin gene expressed by this cell having changed in different lineages; photoreceptors might, at one point, have coexpressed both blue-sensitive ciliary and rhabdomeric opsins. This possibility is not without precedent, as both vertebrate and invertebrate photoreceptors are known to coexpress different opsin genes with similar spectral sensitivities. In some cases, opsin genes with similar spectral sensitivity but which are nonetheless highly divergent at the molecular level are coexpressed, such as the blue-sensitive melanopsin and retinal cone opsins of the chick retina. Analysis of Spalt family gene expression in invertebrates from multiple phyla with well-characterized color vision should further clarify this finding (de Melo, 2011).

Sall3 also plays a pivotal role in regulating the differentiation of horizontal cells, one of two cell types directly postsynaptic to cone photoreceptors, and might do so in part through maintenance of Lhx1 expression. No defects were observed in the expression of other previously reported horizontal cell-expressed transcription factors in Sall3-/- retinas, including Pax6, Foxn4 and Ptf1a, although cell counts did reveal a reduction in the number of Prox1-positive cells in the dorsal retina at P0. However, the final laminar position of Sall3-/- horizontal cells closely resembles that seen in Lhx1 mutants. Horizontal cells of Chx10-Cre; Lhx1lox/lox mutants fail to undergo initial outward radial migration, resulting in ectopic localization to the inner INL and extension of their dendritic arbor within the IPL. Horizontal cells of Sall3-/- retinas appear to initiate Lhx1 expression and undergo outward radial migration normally. However, by P0, Lhx1 expression is drastically reduced, and at later ages the majority of horizontal cell bodies are found at the scleral border of the IPL, eventually taking up residence with amacrine cells and extending dendrites into the IPL, phenocopying the Lhx1 mutants. The phenomenon of ectopic inner nuclear/plexiform layer-associated horizontal cells was also seen in Sall3 overexpression experiments in which Sall3 appeared to be sufficient to at least partially specify horizontal cells, including activating Prox1 and NF165. The lack of coexpression with markers specific to AII amacrine cells indicated that the Prox1-expressing wide-field cells generated in Sall3 electroporation experiments were not a dysmorphic AII population. This does not exclude the possibility that Sall3 overexpression results in the generation of a rare Sall3+ Prox1+ wide-field amacrine subclass. Notably, Spalt genes also regulate prospero expression in developing Drosophila photoreceptors, which then acts to guide differentiation of the R7 photoreceptor subtype. Overexpression of Sall3 in neonatal retinas was not sufficient to induce expression of Lhx1, and, consequently, the horizontal-like cells demonstrated the same ectopic positioning seen in Lhx1 and Sall3 mutants (de Melo, 2011).

The crucial role of Sall3 in regulating Lhx1 expression is further underlined by microarray analysis, which indicates that Lhx1 is one of the most strongly downregulated genes in Sall3-/- retinas. Taken together, these data suggest that sustained Lhx1 expression might regulate multiple stages of horizontal cell migration, and that Sall3 is necessary to maintain Lhx1 expression during the postnatal differentiation of horizontal cells. A limited number of Sall3+ NF165+ and Sall3+ Prox1+ horizontal cells are present in Six3-Cre; Lhx1lox/lox retinas, implying that Sall3 regulates aspects of horizontal cell development at least in part through an Lhx1-independent pathway and that expression of Sall3 itself might not require Lhx1 (de Melo, 2011).

A subset of bipolar interneurons selectively expressed Sall3. Dedicated S-cone-selective bipolar cells in the mouse retina that are analogous to primate S-cone-selective midget bipolar cells have been identified. The tantalizing possibility exists that Sall3 might regulate the differentiation of S-cones and their dedicated bipolar interneuron. However, the microarray analysis did not reveal any significant changes in known mouse bipolar-expressed genes, and the role of Sall3 in bipolar interneuron development remains to be resolved (de Melo, 2011).

Mutation of SALL2 causes recessive ocular coloboma in humans and mice

Ocular coloboma is a congenital defect resulting from failure of normal closure of the optic fissure during embryonic eye development. This birth defect causes childhood blindness worldwide, yet the genetic etiology is poorly understood. This study identified a novel homozygous mutation in the SALL2 gene in members of a consanguineous family affected with non-syndromic ocular coloboma variably affecting the iris and retina. This mutation, c.85G>T, introduces a premature termination codon (p.Glu29*) predicted to truncate the SALL2 protein so that it lacks three clusters of zinc-finger motifs that are essential for DNA-binding activity. This discovery identifies SALL2 as the third member of the Drosophila homeotic Spalt-like family of developmental transcription factor genes implicated in human disease. SALL2 is expressed in the developing human retina at the time of, and subsequent to, optic fissure closure. Analysis of Sall2-deficient mouse embryos revealed delayed apposition of the optic fissure margins and the persistence of an anterior retinal coloboma phenotype after birth. Sall2-deficient embryos displayed correct posterior closure toward the optic nerve head, and upon contact of the fissure margins, dissolution of the basal lamina occurred and PAX2, known to be critical for this process, was expressed normally. Anterior closure was disrupted with the fissure margins failing to meet, or in some cases misaligning leading to a retinal lesion. These observations demonstrate, for the first time, a role for SALL2 in eye morphogenesis and that loss of function of the gene causes ocular coloboma in humans and mice (Kelberman, 2014).

The transcription factor SALL4 regulates stemness of EpCAM-positive hepatocellular carcinoma

Recent evidence suggests that hepatocellular carcinoma can be classified into certain molecular subtypes with distinct prognoses based on the stem/maturational status of the tumor. This study investigated the transcription program deregulated in hepatocellular carcinomas with stem cell features. Gene and protein expression profiles were obtained from 238 (analyzed by microarray), 144 (analyzed by immunohistochemistry), and 61 (analyzed by qRT-PCR) hepatocellular carcinoma cases. Activation/suppression of an identified transcription factor was used to evaluate its role in cell lines. The relationship of the transcription factor and prognosis was statistically examined. The transcription factor SALL4, known to regulate stemness in embryonic and hematopoietic stem cells, was found to be activated in a hepatocellular carcinoma subtype with stem cell features. SALL4-positive hepatocellular carcinoma patients were associated with high values of serum alpha fetoprotein, high frequency of hepatitis B virus infection, and poor prognosis after surgery compared with SALL4-negative patients. Activation of SALL4 enhanced spheroid formation and invasion capacities, key characteristics of cancer stem cells, and up-regulated the hepatic stem cell markers KRT19, EPCAM, and CD44 in cell lines. Knockdown of SALL4 resulted in the down-regulation of these stem cell markers, together with attenuation of the invasion capacity. The SALL4 expression status was associated with histone deacetylase activity in cell lines, and the histone deacetylase inhibitor successfully suppressed proliferation of SALL4-positive hepatocellular carcinoma cells. It is concluded that SALL4 is a valuable biomarker and therapeutic target for the diagnosis and treatment of hepatocellular carcinoma with stem cell features (Zheng, 2014).

Sall4 controls differentiation of pluripotent cells independently of the Nucleosome Remodelling and Deacetylation (NuRD) complex

Sall4 is an essential transcription factor for early mammalian development and is frequently overexpressed in cancer. Though it is reported to play an important role in embryonic stem cell self-renewal, whether it is an essential pluripotency factor has been disputed. This study shows that Sall4 is dispensable for ES cell pluripotency. Sall4 is an enhancer-binding protein that prevents precocious activation of the neural gene expression program in ES cells but is not required for maintenance of the pluripotency gene regulatory network. While a proportion of Sall4 protein physically associates with the Nucleosome Remodeling and Deacetylase (NuRD) complex, Sall4 neither recruits NuRD to chromatin nor influences transcription via NuRD; rather free Sall4 protein regulates transcription independently of NuRD. A model is proposed whereby enhancer binding by Sall4 and other pluripotency-associated transcription factors is responsible for maintaining the balance between transcriptional programs in pluripotent cells (Miller, 2016).

spalt: Biological Overview | Regulation | Targets of Activity | Developmental Biology | Effects of Mutation | References

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