Nuclear adaptor Ldb1 regulates a transcriptional program essential for the maintenance of hematopoietic stem cells.

The nuclear adaptor Ldb1 functions as a core component of multiprotein transcription complexes that regulate differentiation in diverse cell types. In the hematopoietic lineage, Ldb1 forms a complex with the non-DNA-binding adaptor Lmo2 and the transcription factors E2A, Scl and GATA-1 (or GATA-2). Here we demonstrate a critical and continuous requirement for Ldb1 in the maintenance of both fetal and adult mouse hematopoietic stem cells (HSCs). Deletion of Ldb1 in hematopoietic progenitors resulted in the downregulation of many transcripts required for HSC maintenance. Genome-wide profiling by chromatin immunoprecipitation followed by sequencing (ChIP-Seq) identified Ldb1 complex-binding sites at highly conserved regions in the promoters of genes involved in HSC maintenance. Our results identify a central role for Ldb1 in regulating the transcriptional program responsible for the maintenance of HSCs.

The stage-dependent roles of Ldb1 and functional redundancy with Ldb2 in mammalian retinogenesis.

The Lim domain-binding proteins are key co-factor proteins that assemble with LIM domains of the LMO/LIM-HD family to form functional complexes that regulate cell proliferation and differentiation. Using conditional mutagenesis and comparative phenotypic analysis, we analyze the function of Ldb1 and Ldb2 in mouse retinal development, and demonstrate overlapping and specific functions of both proteins. Ldb1 interacts with Lhx2 in the embryonic retina and both Ldb1 and Ldb2 play a key role in maintaining the pool of retinal progenitor cells. This is accomplished by controlling the expression of the Vsx2 and Rax, and components of the Notch and Hedgehog signaling pathways. Furthermore, the Ldb1/Ldb2-mediated complex is essential for generation of early-born photoreceptors through the regulation of Rax and Crx. Finally, we demonstrate functional redundancy between Ldb1 and Ldb2. Ldb1 can fully compensate the loss of Ldb2 during all phases of retinal development, whereas Ldb2 alone is sufficient to sustain activity of Lhx2 in both early- and late-stage RPCs and in Müller glia. By contrast, loss of Ldb1 disrupts activity of the LIM domain factors in neuronal precursors. An intricate regulatory network exists that is mediated by Ldb1 and Ldb2, and promotes RPC proliferation and multipotency; it also controls specification of mammalian retina cells.

Compensatory Actions of Ldb Adaptor Proteins During Corticospinal Motor Neuron Differentiation.

Although many genes that specify neocortical projection neuron subtypes have been identified, the downstream effectors that control differentiation of those subtypes remain largely unknown. Here, we demonstrate that the LIM domain-binding proteins Ldb1 and Ldb2 exhibit dynamic and inversely correlated expression patterns during cerebral cortical development. Ldb1-deficient brains display severe defects in proliferation and changes in regionalization, phenotypes resembling those of Lhx mutants. Ldb2-deficient brains, on the other hand, exhibit striking phenotypes affecting layer 5 pyramidal neurons: Immature neurons have an impaired capacity to segregate into mature callosal and subcerebral projection neurons. The analysis of Ldb2 single-mutant mice reveals a compensatory role of Ldb1 for Ldb2 during corticospinal motor neuron (CSMN) differentiation. Animals lacking both Ldb1 and Ldb2 uncover the requirement for Ldb2 during CSMN differentiation, manifested as incomplete CSMN differentiation, and ultimately leading to a failure of the corticospinal tract.

Ldb1 is essential for development of Nkx2.1 lineage derived GABAergic and cholinergic neurons in the telencephalon.

The progenitor zones of the embryonic mouse ventral telencephalon give rise to GABAergic and cholinergic neurons. We have shown previously that two LIM-homeodomain (LIM-HD) transcription factors, Lhx6 and Lhx8, that are downstream of Nkx2.1, are critical for the development of telencephalic GABAergic and cholinergic neurons. Here we investigate the role of Ldb1, a nuclear protein that binds directly to all LIM-HD factors, in the development of these ventral telencephalon derived neurons. We show that Ldb1 is expressed in the Nkx2.1 cell lineage during embryonic development and in mature neurons. Conditional deletion of Ldb1 causes defects in the expression of a series of genes in the ventral telencephalon and severe impairment in the tangential migration of cortical interneurons from the ventral telencephalon. Similar to the phenotypes observed in Lhx6 or Lhx8 mutant mice, the Ldb1 conditional mutants show a reduction in the number of both GABAergic and cholinergic neurons in the telencephalon. Furthermore, our analysis reveals defects in the development of the parvalbumin-positive neurons in the globus pallidus and striatum of the Ldb1 mutants. These results provide evidence that Ldb1 plays an essential role as a transcription co-regulator of Lhx6 and Lhx8 in the control of mammalian telencephalon development.

Stringent requirement of a proper level of canonical WNT signalling activity for head formation in mouse embryo.

In mouse embryos, loss of Dickkopf-1 (DKK1) activity is associated with an ectopic activation of WNT signalling responses in the precursors of the craniofacial structures and leads to a complete truncation of the head at early organogenesis. Here, we show that ENU-induced mutations of genes coding for two WNT canonical pathway factors, the co-receptor LRP6 and the transcriptional co-activator ß-catenin, also elicit an ectopic signalling response and result in loss of the rostral tissues of the forebrain. Compound mutant embryos harbouring combinations of mutant alleles of Lrp6, Ctnnb1 and Dkk1 recapitulate the partial to complete head truncation phenotype of individual homozygous mutants. The demonstration of a synergistic interaction of Dkk1, Lrp6 and Ctnnb1 provides compelling evidence supporting the concepts that (1) stringent regulation of the level of canonical WNT signalling is necessary for head formation, (2) activity of the canonical pathway is sufficient to account for the phenotypic effects of mutations in three different components of the signal cascade and (3) rostral parts of the brain and the head are differentially more sensitive to canonical WNT signalling and their development is contingent on negative modulation of WNT signalling activity.

Transcriptional regulation by CHIP/LDB complexes.

It is increasingly clear that transcription factors play versatile roles in turning genes "on" or "off" depending on cellular context via the various transcription complexes they form. This poses a major challenge in unraveling combinatorial transcription complex codes. Here we use the powerful genetics of Drosophila combined with microarray and bioinformatics analyses to tackle this challenge. The nuclear adaptor CHIP/LDB is a major developmental regulator capable of forming tissue-specific transcription complexes with various types of transcription factors and cofactors, making it a valuable model to study the intricacies of gene regulation. To date only few CHIP/LDB complexes target genes have been identified, and possible tissue-dependent crosstalk between these complexes has not been rigorously explored. SSDP proteins protect CHIP/LDB complexes from proteasome dependent degradation and are rate-limiting cofactors for these complexes. By using mutations in SSDP, we identified 189 down-stream targets of CHIP/LDB and show that these genes are enriched for the binding sites of APTEROUS (AP) and PANNIER (PNR), two well studied transcription factors associated with CHIP/LDB complexes. We performed extensive genetic screens and identified target genes that genetically interact with components of CHIP/LDB complexes in directing the development of the wings (28 genes) and thoracic bristles (23 genes). Moreover, by in vivo RNAi silencing we uncovered novel roles for two of the target genes, xbp1 and Gs-alpha, in early development of these structures. Taken together, our results suggest that loss of SSDP disrupts the normal balance between the CHIP-AP and the CHIP-PNR transcription complexes, resulting in down-regulation of CHIP-AP target genes and the concomitant up-regulation of CHIP-PNR target genes. Understanding the combinatorial nature of transcription complexes as presented here is crucial to the study of transcription regulation of gene batteries required for development.

Isl1 and Ldb co-regulators of transcription are essential early determinants of mouse limb development.

BACKGROUND: The developing limb has served as an excellent model for studying pattern formation and signal transduction in mammalians. Many of the crucial genes that regulate growth and patterning of the limb following limb bud formation are now well known. However, details regarding the control of limb initiation and early stages of outgrowth remain to be defined. This report is focused on genetic events that pave the way for the establishment of a hindlimb bud. RESULTS: Fgf10 and Tbx are crucial for early phases of limb bud initiation. Here we show that in the absence of Isl1 or of Ldb1/2, there is no hindlimb bud development. Fgf10 expression in the bud mesenchyme is dependent on Isl1 and its Ldb co-regulators. CONCLUSIONS: Thus, Isl1 and the Ldb co-regulators of transcription are essential early determinants of mouse limb development. Isl1/Ldb complexes regulate Fgf10 to orchestrate the earliest stages of hindlimb formation.

Cleft palate defect of Dlx1/2-/- mutant mice is caused by lack of vertical outgrowth in the posterior palate.

BACKGROUND: Mice lacking the activities of Dlx1 and Dlx2 (Dlx1/2-/-) exhibit cleft palate, one of the most common human congenital defects, but the etiology behind this phenotype has been unknown. Therefore, we analyzed the morphological, cellular, and molecular changes caused by inactivation of Dlx1 and Dlx2 as related to palate development. RESULTS: Dlx1/2-/- mutants exhibited lack of vertical growth in the posterior palate during the earliest stage of palatogenesis. We attributed this growth deficiency to reduced cell proliferation. Expression of a cell cycle regulator Ccnd1 was specifically down-regulated in the same region. Previous studies established that the epithelial-mesenchymal signaling loop involving Shh, Bmp4, and Fgf10 is important for cell proliferation and tissue growth during palate development. This signaling loop was disrupted in Dlx1/2-/- palate. Interestingly, however, the decreases in Ccnd1 expression and mitosis in Dlx1/2-/- mutants were independent of this signaling loop. Finally, Dlx1/2 activity was required for normal expression of several transcription factor genes whose mutation results in palate defects. CONCLUSIONS: The functions of Dlx1 and Dlx2 are crucial for the initial formation of the posterior palatal shelves, and that the Dlx genes lie upstream of multiple signaling molecules and transcription factors important for later stages of palatogenesis.

LIM-homeobox gene Lhx5 is required for normal development of Cajal-Retzius cells.

Cajal-Retzius (C-R) cells play important roles in the lamination of the mammalian cortex via reelin secretion. The genetic mechanisms underlying the development of these neurons have just begun to be unraveled. Here, we show that two closely related LIM-homeobox genes Lhx1 and Lhx5 are expressed in reelin+ cells in various regions in the mouse telencephalon at or adjacent to sites where the C-R cells are generated, including the cortical hem, the mantle region of the septal/retrobulbar area, and the ventral pallium. Whereas Lhx5 is expressed in all of these reelin-expressing domains, Lhx1 is preferentially expressed in the septal area and in a continuous domain spanning from lateral olfactory region to caudomedial territories. Genetic ablation of Lhx5 results in decreased reelin+ and p73+ cells in the neocortical anlage, in the cortical hem, and in the septal, olfactory, and caudomedial telencephalic regions. The overall reduction in number of C-R cells in Lhx5 mutants is accompanied by formation of ectopic reelin+ cell clusters at the caudal telencephalon. Based on differential expression of molecular markers and by fluorescent cell tracing in cultured embryos, we located the origin of reelin+ ectopic cell clusters at the caudomedial telencephalic region. We also confirmed the existence of a normal migration stream of reelin+ cells from the caudomedial area to telencephalic olfactory territories in wild-type embryos. These results reveal a complex role for Lhx5 in regulating the development and normal distribution of C-R cells in the developing forebrain.

A role of the LIM-homeobox gene Lhx2 in the regulation of pituitary development.

The mammalian pituitary gland originates from two separate germinal tissues during embryonic development. The anterior and intermediate lobes of the pituitary are derived from Rathke's pouch, a pocket formed by an invagination of the oral ectoderm. The posterior lobe is derived from the infundibulum, which is formed by evagination of the neuroectoderm in the ventral diencephalon. Previous studies have shown that development of Rathke's pouch and the generation of distinct populations of hormone-producing endocrine cell lineages in the anterior/intermediate pituitary lobes is regulated by a number of transcription factors expressed in the pouch and by inductive signals from the ventral diencephalon/infundibulum. However, little is known about factors that regulate the development of the posterior pituitary lobe. In this study, we show that the LIM-homeobox gene Lhx2 is extensively expressed in the developing ventral diencephalon, including the infundibulum and the posterior lobe of the pituitary. Deletion of Lhx2 gene results in persistent cell proliferation, a complete failure of evagination of the neuroectoderm in the ventral diencephalon, and defects in the formation of the distinct morphological features of the infundibulum and the posterior pituitary lobe. Rathke's pouch is formed and endocrine cell lineages are generated in the anterior/intermediate pituitary lobes of the Lhx2 mutant. However, the shape and organization of the pouch and the anterior/intermediate pituitary lobes are severely altered due to the defects in development of the infundibulum and the posterior lobe. Our study thus reveals an essential role for Lhx2 in the regulation of posterior pituitary development and suggests a mechanism whereby development of the posterior lobe may affect the development of the anterior and intermediate lobes of the pituitary gland.

The Rho guanine nucleotide exchange factor AKAP13 (BRX) is essential for cardiac development in mice.

A fundamental biologic principle is that diverse biologic signals are channeled through shared signaling cascades to regulate development. Large scaffold proteins that bind multiple proteins are capable of coordinating shared signaling pathways to provide specificity to activation of key developmental genes. Although much is known about transcription factors and target genes that regulate cardiomyocyte differentiation, less is known about scaffold proteins that couple signals at the cell surface to differentiation factors in developing heart cells. Here we show that AKAP13 (also known as Brx-1, AKAP-Lbc, and proto-Lbc), a unique protein kinase A-anchoring protein (AKAP) guanine nucleotide exchange region belonging to the Dbl family of oncogenes, is essential for cardiac development. Cardiomyocytes of Akap13-null mice had deficient sarcomere formation, and developing hearts were thin-walled and mice died at embryonic day 10.5-11.0. Disruption of Akap13 was accompanied by reduced expression of Mef2C. Consistent with a role of AKAP13 upstream of MEF2C, Akap13 siRNA led to a reduction in Mef2C mRNA, and overexpression of AKAP13 augmented MEF2C-dependent reporter activity. The results suggest that AKAP13 coordinates Galpha(12) and Rho signaling to an essential transcription program in developing cardiomyocytes.

Intraflagellar transport protein 172 is essential for primary cilia formation and plays a vital role in patterning the mammalian brain.

IFT172, also known as Selective Lim-domain Binding protein (SLB), is a component of the intraflagellar transport (IFT) complex. In order to evaluate the biological role of the Ift172 gene, we generated a loss-of-function mutation in the mouse. The resulting Slb mutant embryos die between E12.5 and 13.0, and exhibit severe cranio-facial malformations, failure to close the cranial neural tube, holoprosencephaly, heart edema and extensive hemorrhages. Cilia outgrowth in cells of the neuroepithelium is initiated but the axonemes are severely truncated and do not contain visible microtubules. Morphological and molecular analyses revealed a global brain-patterning defect along the dorsal-ventral (DV) and anterior-posterior (AP) axes. We demonstrate that Ift172 gene function is required for early regulation of Fgf8 at the midbrain-hindbrain boundary and maintenance of the isthmic organizer. In addition, Ift172 is required for proper function of the embryonic node, the early embryonic organizer and for formation of the head organizing center (the anterior mesendoderm, or AME). We propose a model suggesting that forebrain and mid-hindbrain growth and AP patterning depends on the early function of Ift172 at gastrulation. Our data suggest that the formation and function of the node and AME in the mouse embryo relies on an indispensable role of Ift172 in cilia morphogenesis and cilia-mediated signaling.

Liver-specific Ldb1 deletion results in enhanced liver cancer development.

BACKGROUND & AIMS: LIM-domain-binding (Ldb) proteins have been demonstrated to be essential not only to key embryonic developmental processes but also to carcinogenesis. We have previously demonstrated Ldb1 to be of high biological and developmental relevance, as a targeted deletion of the Ldb1 gene in mice results in an embryonic lethal and pleiotropic phenotype. METHODS: We have now established a liver-specific Ldb1 knock out to investigate the role of Ldb1 in carcinogenesis, in particular in hepatocellular carcinoma (HCC) development, in vivo. RESULTS: These mice demonstrated a significantly enhanced growth of liver cancer by means of tumor size and number, advocating for an essential role of Ldb1 in HCC development. In addition, proliferation and resistance against apoptosis were increased. In order to identify the functional disturbances due to a lack of Ldb1, we performed a 15k mouse gene microarray expression analysis. We found the Myc oncogene to be regulated in the microarray analysis and were able to further confirm this regulation by demonstrating an over-expression of its downstream target Cyclin D1. Furthermore, we were able to demonstrate a down-regulation of the tumor suppressor p21. Finally, the liver stem cell marker EpCAM was also identified to be over expressed in Ldb1(-/-) knock out mice. CONCLUSIONS: We have established a significant role of Ldb1 in cancer development. Furthermore, we provided evidence for a myc/cyclin D1, p21, and EpCAM-dependent signalling to be key downstream regulators of this novel concept in HCC development.

Impaired neutrophil activity and increased susceptibility to bacterial infection in mice lacking glucose-6-phosphatase-beta.

Neutropenia and neutrophil dysfunction are common in many diseases, although their etiology is often unclear. Previous views held that there was a single ER enzyme, glucose-6-phosphatase-alpha (G6Pase-alpha), whose activity--limited to the liver, kidney, and intestine--was solely responsible for the final stages of gluconeogenesis and glycogenolysis, in which glucose-6-phosphate (G6P) is hydrolyzed to glucose for release to the blood. Recently, we characterized a second G6Pase activity, that of G6Pase-beta (also known as G6PC), which is also capable of hydrolyzing G6P to glucose but is ubiquitously expressed and not implicated in interprandial blood glucose homeostasis. We now report that the absence of G6Pase-beta led to neutropenia; defects in neutrophil respiratory burst, chemotaxis, and calcium flux; and increased susceptibility to bacterial infection. Consistent with this, G6Pase-beta-deficient (G6pc3-/-) mice with experimental peritonitis exhibited increased expression of the glucose-regulated proteins upregulated during ER stress in their neutrophils and bone marrow, and the G6pc3-/- neutrophils exhibited an enhanced rate of apoptosis. Our results define a molecular pathway to neutropenia and neutrophil dysfunction of previously unknown etiology, providing a potential model for the treatment of these conditions.

Generation of mice with a conditional allele for G6pc.

Glucose-6-phosphatase-alpha (G6Pase-alpha or G6PC) catalyzes the hydrolysis of glucose-6-phosphate to glucose and is a key enzyme in interprandial glucose homeostasis. Mutations in the human G6PC gene, expressed primarily in the liver, kidney, and intestine, cause glycogen storage disease Type Ia (GSD-Ia), an autosomal recessive disorder characterized by a disturbed glucose homeostasis. For better understanding of the roles of G6Pase-alpha in different tissues and in pathological conditions, we have generated mice harboring a conditional null allele for G6pc by flanking Exon 3 of the G6pc gene with loxP sites. We confirmed the null phenotype by using the EIIa-Cre transgenic approach to generate mice lacking Exon 3 of the G6pc gene. The resulting homozygous Cre-recombined null mice manifest a phenotype mimicking G6Pase-alpha-deficient mice and human GSD-Ia patients. This G6pc conditional null allele will be valuable to examine the consequence of tissue-specific G6Pase-alpha deficiency and the mechanisms of long-term complications in GSD-Ia.

The neuronal differentiation potential of Ldb1-null mutant embryonic stem cells is dependent on extrinsic influences.

LIM-domain binding protein 1 (Ldb1) is a multiadaptor protein that mediates the action of transcription factors, including LIM-homeodomain proteins. To elucidate the functional role of Ldb1 in the neuronal differentiation of embryonic stem (ES) cells, we have generated Ldb1-null mutant (Ldb1-/-) ES cells and examined neuronal differentiation potentials in vitro using two different neuronal differentiation protocols. When subjected to a five-stage protocol that recapitulates in vivo conditions of neuronal differentiation, wild-type ES cells differentiated into a wide spectrum of neuronal cell types. However, Ldb1-/- ES cells did not differentiate into neuronal cells; instead, they differentiated into sarcomeric alpha-actinin-positive muscle cells. In contrast, when an adherent monolayer culture procedure (which is based on the default mechanism of neural induction and eliminates environmental influences) was applied, both wild-type and Ldb1-/- ES cells differentiated into MAP2-positive mature neurons. Comparison of the results obtained when two different neuronal differentiation protocols were used suggests that Ldb1-/- ES cells have an innate potential to differentiate into neuronal cells, but this potential can be inhibited by environmental influences. Disclosure of potential conflicts of interest is found at the end of this article.

Generalized tetracycline induced Cre recombinase expression through the ROSA26 locus of recombinant mice.

Inducible Cre recombinase systems have been developed to bypass initial lethal phenotypes and to provide access to later embryonic or adult phenotypes. Here we describe the generation of a recombinant mouse that combines a tetracycline dependent switch with generalized Cre recombinase expression by targeting the ubiquitously expressed ROSA26 locus. This transgenic strain was developed using a simplified gene delivery system integrating both elements, the reverse tetracycline controlled trans-activator (rtTA) and rtTA inducible promoter into a single vector. In this transgenic strain, the endogenous ROSA26 promoter drives rtTA expression through a splice acceptor site. The tetracycline inducible promoter, cloned in opposite orientation to the ROSA26 locus and separated from the rtTA element by a 5 kb human p53 intron, drives Cre recombinase expression. Crossing these mice with a Cre reporter strain showed that Cre DNA-mediated recombination was ubiquitously and effectively induced during various prenatal developmental windows. Background Cre recombinase expression levels were observed in some tissues in the absence of the inducer, mostly during late embryonic developmental stages and in adult animals. Background recombination levels were low during development and most prominent in nervous tissue. Cre recombinase expression could not be effectively induced in adult animals. While rtTA mRNA levels were high in developmental and adult tissues, Cre recombinase mRNA levels remained low after doxycycline treatment. The mouse strain described here provides a valuable tool to further analyze the function of genes during specific developmental windows, by allowing the effective inactivation of their function throughout defined stages of embryonic development.

Generation and characterization of dickkopf3 mutant mice.

dickkopf (dkk) genes encode a small family of secreted Wnt antagonists, except for dkk3, which is divergent and whose function is poorly understood. Here, we describe the generation and characterization of dkk3 mutant mice. dkk3-deficient mice are viable and fertile. Phenotypic analysis shows no major alterations in organ morphology, physiology, and most clinical chemistry parameters. Since Dkk3 was proposed to function as thyroid hormone binding protein, we have analyzed deiodinase activities, as well as thyroid hormone levels. Mutant mice are euthyroid, and the data do not support a relationship of dkk3 with thyroid hormone metabolism. Altered phenotypes in dkk3 mutant mice were observed in the frequency of NK cells, immunoglobulin M, hemoglobin, and hematocrit levels, as well as lung ventilation. Furthermore, dkk3-deficient mice display hyperactivity.

Multidrug resistance protein 4 protects bone marrow, thymus, spleen, and intestine from nucleotide analogue-induced damage.

Nucleoside-based analogues are mainstays in the treatment of cancer, viral infections, and inflammatory diseases. Recent studies showing that the ATP-binding cassette transporter, multidrug resistance protein 4, is able to efflux nucleoside and nucleotide analogues from transfected cells suggests that the pump may affect the efficacy of this class of agents. However, the in vivo pharmacologic functions of the pump are largely unexplored. Here, using Mrp4(-/-) mice as a model system, and the nucleotide analogue, 9'-(2'-phosphonylmethoxyethyl)-adenine (PMEA) as a probe, we investigate the ability of Mrp4 to function in vivo as an endogenous resistance factor. In the absence of alterations in plasma PMEA levels, Mrp4-null mice treated with PMEA exhibit increased lethality associated with marked toxicity in several tissues. Affected tissues include the bone marrow, spleen, thymus, and gastrointestinal tract. In addition, PMEA penetration into the brain is increased in Mrp4(-/-) mice. These findings indicate that Mrp4 is an endogenous resistance factor, and that the pump may be a component of the blood-brain barrier for nucleoside-based analogues. This is the first demonstration that an ATP-binding cassette transporter can affect in vivo tissue sensitivity towards this class of agents.

Tyrosine phosphorylation controls nuclear localization and transcriptional activity of Ssdp1 in mammalian cells.

The LIM-HD proteins interact with different cofactors, including Ssdp1 to regulate development in a diverse range of species. The single stranded DNA binding protein (Ssdp1) is a member of an evolutionarily conserved family of proteins that regulate critical transcriptional processes during embryonic development. Ssdp1 is localized predominantly in the cytoplasm of 293T cells but is translocated to the nucleus when co-transfected with Lck, a member of the Src family of non-receptor tyrosine kinases. The Src tyrosine kinase inhibitor PP2 blocked the nuclear translocation of Ssdp1. Western blot analysis showed that co-expression of Ssdp1 and Lck in 293T cells induces Ssdp1 phosphorylation. Mutation of the Ssdp1 N terminal tyrosine residues 23 and 25 markedly reduced both the phosphorylation and the nuclear localization of Ssdp1. Lck enhanced the transcriptional activity of Ssdp1 in the context of known components of a LIM-homeodomain (LIM-HD)/cofactor complex. We propose that phosphorylation involving N-terminal tyrosine residues of Ssdp1 is a means of regulating its nuclear localization and subsequent transcriptional activation of LIM-HD complexes.