LIM-homeodomain Transcription Factor Lmx1b Research Articles

LC3B is a cofactor for LMX1B-mediated transcription of autophagy genes in dopaminergic neurons.

It is becoming increasingly clear that the Atg8 family of autophagy proteins have roles not only in the cytoplasm, but also in the cell nucleus. In this issue, Jiménez-Moreno et al. (2023. J. Cell Biol.https://doi.org/10.1083/jcb.201910133) report that nuclear LC3B binds to the LIM homeodomain transcription factor LMX1B and acts as a cofactor for LMX1B-mediated transcription of autophagy genes, providing stress protection and ensuring survival of midbrain dopaminergic neurons.

Transcription Factor Lmx1b Negatively Regulates Osteoblast Differentiation and Bone Formation.

The LIM-homeodomain transcription factor Lmx1b plays a key role in body pattern formation during development. Although Lmx1b is essential for the normal development of multiple tissues, its regulatory mechanism in bone cells remains unclear. Here, we demonstrated that Lmx1b negatively regulates bone morphogenic protein 2 (BMP2)-induced osteoblast differentiation. Overexpressed Lmx1b in the osteoblast precursor cells inhibited alkaline phosphatase (ALP) activity and nodule formation, as well as the expression of osteoblast maker genes, including runt-related transcription factor 2 (Runx2), alkaline phosphatase (Alpl), bone sialoprotein (Ibsp), and osteocalcin (Bglap). Conversely, the knockdown of Lmx1b in the osteoblast precursors enhanced the osteoblast differentiation and function. Lmx1b physically interacted with and repressed the transcriptional activity of Runx2 by reducing the recruitment of Runx2 to the promoter region of its target genes. In vivo analysis of BMP2-induced ectopic bone formation revealed that the knockdown of Lmx1b promoted osteogenic differentiation and bone regeneration. Our data demonstrate that Lmx1b negatively regulates osteoblast differentiation and function through regulation of Runx2 and provides a molecular basis for therapeutic targets for bone diseases.

Nail-Patella syndrome with early onset end-stage renal disease in a child with a novel heterozygous missense mutation in the LMX1B homeodomain: A case report.

Nail-Patella syndrome (NPS) is an inherited disease characterized by nail and skeletal anomalies, nephropathy and glaucoma. The diagnosis of NPS is based on clinical findings, including hypoplastic or absent patella, dystrophic nails, dysplasia of the elbows and iliac horns. However, the main determinant of NPS prognosis is nephropathy, which may range from asymptomatic proteinuria to end-stage renal disease. NPS is caused by heterozygous loss-of-function mutations in the LMX1B gene, which encodes the LIM homeodomain transcription factor LMX1B. LMX1B serves an essential role in the physiological development of dorsal-ventral limb structures, morphogenesis and function of podocytes, as well as in development of the anterior segments of the eyes, and in certain types of neurons. The present study aimed to identify the disease-causing mutation in a 2-year old girl with nephrotic syndrome that evolved rapidly to end-stage renal disease. The patient showed classical symptoms of NPS including dystrophic nails and an absence of the patellae. DNA sequence analysis identified a novel missense variant in exon 4 of LMX1B (c.709T>C, p.S237P); this substitution affected a conserved serine residue in the homeodomain of LMX1B and was predicted to be pathogenic. In silico modeling of the homeodomain revealed that the p.S237P mutation converted the A236-S237-F238 segment of α-helix 1 into a strand. It was hypothesized that this mutation affected binding of the transcription factor to its target DNA, thus abrogating transcription activation, which would explain the phenotype that manifested in the patient.

Novel missense mutation affecting the LIM-A domain of LMX1B in a family with Nail-Patella syndrome.

Nail-patella syndrome (NPS) is a rare autosomal dominant disease characterized by developmental defects of dorsal limb structures, the kidney, and the eye, that manifest as dysplastic nails, hypoplastic or absent patella, elbow dysplasia, iliac horns, glomerulopathy, and adult-onset glaucoma, respectively. This disorder is inherited in an autosomal dominant mode and is caused by heterozygous loss-of-function mutations in the LMX1B gene, which encodes the LIM homeodomain transcription factor LMX1B. In this study, we report the clinical findings of a Spanish family, from the Canary Islands, with three affected members who displayed varying phenotypes. DNA sequence analysis identified a novel heterozygous missense mutation in LMX1B, c.305A>G, p.(Y102C), that segregated with the disease. The tyrosine residue affected by the mutation is highly conserved in evolution, and is located in the LIM-A domain, next to one of the cysteine residues involved in zinc binding, suggesting that p.(Y102C) affects LMX1B function by disturbing its interactions with other proteins. Our results expand the mutation spectrum of LMX1B and provide insight into the molecular mechanisms of NPS pathology.

Anti-osteogenic function of a LIM-homeodomain transcription factor LMX1B is essential to early patterning of the calvaria

The calvaria (upper part of the skull) is made of plates of bone and fibrous joints (sutures and fontanelles), and the proper balance and organization of these components are crucial to normal development of the calvaria. In a mouse embryo, the calvaria develops from a layer of head mesenchyme that surrounds the brain from shortly after mid-gestation. The mesenchyme just above the eye (supra-orbital mesenchyme, SOM) generates ossification centers for the bones, which then grow toward the apex gradually. In contrast, the mesenchyme apical to SOM (early migrating mesenchyme, EMM), including the area at the vertex, does not generate an ossification center. As a result, the dorsal midline of the head is occupied by sutures and fontanelles at birth. To date, the molecular basis for this regional difference in developmental programs is unknown.The current study provides vital insights into the genetic regulation of calvarial patterning. First, we showed that osteogenic signals were active in both EMM and SOM during normal development, which suggested the presence of an anti-osteogenic factor in EMM to counter the effect of these signals. Subsequently, we identified Lmx1b as an anti-osteogenic gene that was expressed in EMM but not in SOM. Furthermore, head mesenchyme-specific deletion of Lmx1b resulted in heterotopic ossification from EMM at the vertex, and craniosynostosis affecting multiple sutures. Conversely, forced expression of Lmx1b in SOM was sufficient to inhibit osteogenic specification. Therefore, we conclude that Lmx1b plays a key role as an anti-osteogenic factor in patterning the head mesenchyme into areas with different osteogenic competence. In turn, this patterning event is crucial to generating the proper organization of the bones and soft tissue joints of the calvaria.

A dominant-negative mutation of mouse Lmx1b causes glaucoma and is semi-lethal via LDB1-mediated dimerization [corrected

Mutations in the LIM-homeodomain transcription factor LMX1B cause nail-patella syndrome, an autosomal dominant pleiotrophic human disorder in which nail, patella and elbow dysplasia is associated with other skeletal abnormalities and variably nephropathy and glaucoma. It is thought to be a haploinsufficient disorder. Studies in the mouse have shown that during development Lmx1b controls limb dorsal-ventral patterning and is also required for kidney and eye development, midbrain-hindbrain boundary establishment and the specification of specific neuronal subtypes. Mice completely deficient for Lmx1b die at birth. In contrast to the situation in humans, heterozygous null mice do not have a mutant phenotype. Here we report a novel mouse mutant Icst, an N-ethyl-N-nitrosourea-induced missense substitution, V265D, in the homeodomain of LMX1B that abolishes DNA binding and thereby the ability to transactivate other genes. Although the homozygous phenotypic consequences of Icst and the null allele of Lmx1b are the same, heterozygous Icst elicits a phenotype whilst the null allele does not. Heterozygous Icst causes glaucomatous eye defects and is semi-lethal, probably due to kidney failure. We show that the null phenotype is rescued more effectively by an Lmx1b transgene than is Icst. Co-immunoprecipitation experiments show that both wild-type and Icst LMX1B are found in complexes with LIM domain binding protein 1 (LDB1), resulting in lower levels of functional LMX1B in Icst heterozygotes than null heterozygotes. We conclude that Icst is a dominant-negative allele of Lmx1b. These findings indicate a reassessment of whether nail-patella syndrome is always haploinsufficient. Furthermore, Icst is a rare example of a model of human glaucoma caused by mutation of the same gene in humans and mice.

LMX1B Is Part of a Transcriptional Complex with PSPC1 and PSF

The LIM homeodomain transcription factor Lmx1b is essential for the development of the isthmic organizer and mesodiencephalic dopaminergic neurons. The uncoupling of Pitx3 and Th expression, in the Lmx1b null mutant, suggests that Lmx1b may act as a positional activator of the mdDA domain, eventually leading to properly differentiating mdDA neurons. In this study, we aimed to elucidate how Lmx1b functions mechanistically in this developmental process, by searching for molecular interactors of Lmx1b at the protein level. Initially, affinity-purification of LMX1B-HIS overexpressed protein in MN9D dopaminergic cells followed by mass-spectrometry analysis, resulted in the identification of PSPC1 protein as a possible binding partner of LMX1B. Subsequent immunoprecipitation experiments revealed an interaction between LMX1B and PSPC1 in a larger protein complex also containing PSF. This complex was observed in vitro and in vivo, and we hypothesize that, via PSF and PSPC1, LMX1B may be part of the previously identified Nurr1 transcriptional complex wherein interaction with the co-repressor PSF and the transcription factor Pitx3 is needed to drive expression of Nurr1 target genes in specifying the dopaminergic phenotype. Furthermore, we identified GRLF1, DHX9, MYO1C, HSP70 and TMPO as potential LMX1B interactors. DHX9 and GRLF1 are highly expressed in the developing mdDA neuronal field, and GRLF1 and MYO1C have both been linked to neurite outgrowth. The identification of these proteins suggests that Lmx1b may act directly in the transcriptional activation of Nurr1 target genes and be involved in other processes like neurite outgrowth as well.

Uncoupling skeletal and connective tissue patterning: conditional deletion in cartilage progenitors reveals cell-autonomous requirements for Lmx1b in dorsal-ventral limb patterning

Integration of muscle, connective tissue and skeletal patterning during development is essential for proper functioning of the musculoskeletal system. How this integration is achieved is poorly understood. There is ample evidence suggesting that skeletal pattern is programmed autonomously, whereas muscle pattern is, for the most part, programmed non-cell-autonomously. Connective tissues depend upon both muscle and skeletal tissues for their proper survival and development. Here, we employed a novel approach to dissect the coordination of musculoskeletal patterning during mouse limb development. Using both conditional gain- and loss-of-function approaches, we selectively deleted or activated the LIM-homeodomain transcription factor Lmx1b in skeletal progenitors using a Sox9-Cre knock-in allele. As Lmx1b is both necessary and sufficient to specify dorsal pattern, this approach allowed us to investigate the effect of selectively deleting or activating Lmx1b in skeletal progenitors on muscle, connective and skeletal tissues during limb development. Our results indicate that whereas Lmx1b activity is required autonomously in skeletal progenitors to direct dorsal pattern, loss or gain of Lmx1b activity in skeletal progenitors has no effect on muscle or connective tissue patterning. Hence, we show for the first time that skeletal and connective tissue patterning can be uncoupled, indicating a degree of autonomy in the formation of the musculoskeletal system.

Postnatal ontogeny of the transcription factor Lmx1b in the mouse central nervous system

The expression profile of Lim homeodomain transcription factor Lmx1b in the mouse brain was investigated at different postnatal stages by immunohistochemistry and in situ hybridization. At postnatal day (P) 7, many Lmx1b-expressing neurons were found in the posterior hypothalamic area, supramammillary nucleus, ventral premammillary nucleus, and subthalamic nucleus. In the midbrain, numerous Lmx1b-expressing neurons were present in the substantia nigra pars compacta and ventral tegmental area. In the hindbrain, Lmx1b-expressing neurons were primarily observed in the raphe nuclei, parabrachial nuclei, principal sensory trigeminal nucleus, nucleus of the solitary tract, and laminae I-II of the medullary dorsal horn as well as spinal dorsal horn. Although expression levels diminished as postnatal life progressed, persistent expression throughout the first year of life was observed in many of these regions. In contrast, Lmx1b was present in a few brain regions (e.g., principal sensory trigeminal nucleus) only in early life with expression expiring by P60. Lmx1b was observed in dopaminergic neurons in the midbrain and serotonergic neurons in the hindbrain, as determined by double labeling with specific markers. In addition, we found that Lmx1b-expressing neurons are not GABAergic, and Lmx1b was colocalized with Tlx3 in the parabrachial nuclei, principal sensory trigeminal nucleus, nucleus of the solitary tract. as well as the medullary and spinal dorsal horns, suggesting that Lmx1b-expressing cells in these areas are excitatory neurons. Our data suggest that Lmx1b is involved in the postnatal maturation of certain types of neurons and maintenance of their normal functions in the adult brain.

Genotype–phenotype studies in nail-patella syndrome show that LMX1B mutation location is involved in the risk of developing nephropathy

Nail-patella syndrome (NPS) is characterized by developmental defects of dorsal limb structures, nephropathy, and glaucoma and is caused by heterozygous mutations in the LIM homeodomain transcription factor LMX1B. In order to identify possible genotype-phenotype correlations, we performed LMX1B mutation analysis and comprehensive investigations of limb, renal, ocular, and audiological characteristics in 106 subjects from 32 NPS families. Remarkable phenotypic variability at the individual, intrafamilial, and interfamilial level was observed for different NPS manifestations. Quantitative urinanalysis revealed proteinuria in 21.3% of individuals. Microalbuminuria was detected in 21.7% of subjects without overt proteinuria. Interestingly, nephropathy appeared significantly more frequent in females. A significant association was established between the presence of clinically relevant renal involvement in an NPS patient and a positive family history of nephropathy. We identified normal-tension glaucoma (NTG) and sensorineural hearing impairment as new symptoms associated with NPS. Sequencing of LMX1B revealed 18 different mutations, including six novel variants, in 28 families. Individuals with an LMX1B mutation located in the homeodomain showed significantly more frequent and higher values of proteinuria compared to subjects carrying mutations in the LIM domains. No clear genotype-phenotype association was apparent for extrarenal manifestations. This is the first study indicating that family history of nephropathy and mutation location might be important in precipitating individual risks for developing NPS renal disease. We suggest that the NPS phenotype is broader than previously described and that NTG and hearing impairment are part of NPS. Further studies on modifier factors are needed to understand the mechanisms underlying phenotypic heterogeneity.

Control of roof plate development and signaling by Lmx1b in the caudal vertebrate CNS.

Numerous studies have identified the roof plate as an important signaling center controlling dorsal interneuron specification and differentiation in the developing spinal cord. Currently, the molecular pathways of roof plate formation and function are poorly understood. We determined that the LIM-homeodomain transcription factor Lmx1b is sufficient to induce functional roof plate in the early chick developing spinal cord. In the chick, Lmx1b acts upstream of Lmx1a in the roof plate developmental program. Once the roof plate forms, we show that Bmp and Wnt signaling are the major components of Lmx1a/b-dependent roof plate dorsal patterning activity. The roof plate function of Lmx1b is not conserved across vertebrates because Lmx1b is not expressed in mouse roof plate progenitors. Instead, mouse caudal CNS roof plate formation relies entirely on Lmx1a. Lmx1b can, however, partially rescue roof plate development in dreher (Lmx1a-/-) mice, indicating that Lmx1b has some functional redundancy to Lmx1a. Furthermore, we demonstrate that the roof plate-inducing activity of Lmx1b can be suppressed by Mash1 (Cash1), which is normally expressed in intermediate neural tube in both chick and mouse. Our data identify Lmx1b as a key regulator of spinal cord roof plate induction and function.

The LIM-homeodomain transcription factor Lmx1b plays a crucial role in podocytes

The LIM-homeodomain transcription factor Lmx1b plays a crucial role in podocytes

The LIM-homeodomain transcription factor Lmx1b plays a crucial role in podocytes

Patients with nail-patella syndrome often suffer from a nephropathy, which ultimately results in chronic renal failure. The finding that this disease is caused by mutations in the transcription factor LMX1B, which in the kidney is expressed exclusively in podocytes, offers the opportunity for a better understanding of the renal pathogenesis. In our analysis of the nephropathy in nail-patella syndrome, we have made use of the Lmx1b knockout mouse. Transmission electron micrographs showed that glomerular development in general and the differentiation of podocytes in particular were severely impaired. The glomerular capillary network was poorly elaborated, fenestrae in the endothelial cells were largely missing, and the glomerular basement membrane was split. In addition podocytes retained a cuboidal shape and did not form foot processes and slit diaphragms. Expression of the α4 chain of collagen IV and of podocin was also severely reduced. Using gel shift assays, we demonstrated that LMX1B bound to two AT-rich sequences in the promoter region of NPHS2, the gene encoding podocin. Our results demonstrate that Lmx1b regulates important steps in glomerular development and establish a link between three hereditary kidney diseases: nail-patella syndrome (Lmx1b), steroid-resistant nephrotic syndrome (podocin), and Alport syndrome (collagen IV α4).

The LIM-homeodomain transcription factor Lmx1b plays a crucial role in podocytes.

Patients with nail-patella syndrome often suffer from a nephropathy, which ultimately results in chronic renal failure. The finding that this disease is caused by mutations in the transcription factor LMX1B, which in the kidney is expressed exclusively in podocytes, offers the opportunity for a better understanding of the renal pathogenesis. In our analysis of the nephropathy in nail-patella syndrome, we have made use of the Lmx1b knockout mouse. Transmission electron micrographs showed that glomerular development in general and the differentiation of podocytes in particular were severely impaired. The glomerular capillary network was poorly elaborated, fenestrae in the endothelial cells were largely missing, and the glomerular basement membrane was split. In addition podocytes retained a cuboidal shape and did not form foot processes and slit diaphragms. Expression of the alpha4 chain of collagen IV and of podocin was also severely reduced. Using gel shift assays, we demonstrated that LMX1B bound to two AT-rich sequences in the promoter region of NPHS2, the gene encoding podocin. Our results demonstrate that Lmx1b regulates important steps in glomerular development and establish a link between three hereditary kidney diseases: nail-patella syndrome (Lmx1b), steroid-resistant nephrotic syndrome (podocin), and Alport syndrome (collagen IV alpha4).

A second independent pathway for development of mesencephalic dopaminergic neurons requires Lmx1b.

We identified the LIM homeodomain transcription factor Lmx1b in the mesencephalic dopamine (mesDA) systems of embryos and adults. Analysis of spatiotemporal expression in Lmx1b null mutants and wild-type mice implicated a cascade involving Lmx1b in the early development of mesDA neurons. Although disruption of this cascade did not block induction of tyrosine hydroxylase (TH), a key enzyme in DA synthesis, or Nurr1, a nuclear hormone receptor, Lmx1b knockout mice failed to induce the mesDA-specific homeodomain gene Ptx3 in TH-positive neurons. Eventually, this small set of TH-positive neurons was lost during embryonic maturation. The data suggest that at least two molecular cascades operate during the specification of the mesDA system, one specifying neurotransmitter phenotype and another essential for other aspects of mesDA neuron differentiation.

Lmx1b, a LIM homeodomain class transcription factor, is necessary for normal development of multiple tissues in the anterior segment of the murine eye

Proper development of the anterior segment of the mammalian eye is critical for normal ocular function. Indeed, several congenital syndromes associated with anterior segment anomalies can lead to impaired vision and glaucoma. One such syndrome is nail patella syndrome (NPS), caused by haploinsufficiency for the LIM-homeodomain transcription factor LMX1B. Although mutations in LMX1B cosegregate with NPS, whether these mutations cause the glaucoma associated with NPS is not known. Here, we provide evidence that the LIM-homeodomain transcription factor lmx1b is an essential regulator of murine anterior segment development. Mice that are homozygous for a targeted mutation of lmx1b display iris and ciliary body hypoplasia, and cornea stromal defects. In addition, two cDNAs normally downregulated in presumptive cornea, mf1 and mfh1, exhibit persistent expression, while keratocan, a keratin sulfate proteoglycan expressed by keratocytes, is not detected in mutant corneas. Moreover, ultrastructural examination of homozygous mutants indicates that corneal collagen fibrillogenesis is perturbed. Taken together, our studies suggest a developmental etiology for glaucoma in NPS patients and highlight lmx1b as an essential regulator of anterior segment morphogenesis and patterning. genesis 26:15-25, 2000.

Simultaneous expression of multi-subunit proteins in mammalian cells using a convenient set of mammalian cell expression vectors.

BioTechniquesVol. 23, No. 3 BenchmarksOpen AccessSimultaneous Expression of Multi-Subunit Proteins in Mammalian Cells Using a Convenient Set of Mammalian Cell Expression VectorsM.I. Cockett, R. Ochalski, K. Benwell, R. Franco & J. Wardwell-SwansonM.I. Cockett*Address correspondence to M.I. Cockett, CNS Disorders, Wyeth-Ayerst Research, CN8000 Princeton, NJ 08543, USA. Internet: E-mail Address: cocketm@war.wyeth.comWyeth-Ayerst Research, Princeton, NJ, USASearch for more papers by this author, R. OchalskiWyeth-Ayerst Research, Princeton, NJ, USASearch for more papers by this author, K. BenwellWyeth-Ayerst Research, Princeton, NJ, USASearch for more papers by this author, R. FrancoWyeth-Ayerst Research, Princeton, NJ, USASearch for more papers by this author & J. Wardwell-SwansonWyeth-Ayerst Research, Princeton, NJ, USASearch for more papers by this authorPublished Online:7 Aug 2018https://doi.org/10.2144/97233bm11AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail FiguresReferencesRelatedDetailsCited ByOn-section correlative light and electron microscopy of large cellular volumes using STEM tomographyThe LIM-homeodomain transcription factor LMX1B regulates expression of NF-kappa B target genesExperimental Cell Research, Vol. 315, No. 1Activation of Serum Response Element by D2 Dopamine Receptor Is Governed by Gβγ-Mediated MAPK and Rho Pathways and Regulated by RGS Proteins18 December 2006 | Pharmacology, Vol. 79, No. 2A Truncated Polycystin-2 Protein Causes Polycystic Kidney Disease and Retinal Degeneration in Transgenic Rats30 August 2006 | Journal of the American Society of Nephrology, Vol. 17, No. 10Analysis of RGSZ1 Protein Interaction with Gαi SubunitsBalanced expression of single subunits in a multisubunit protein, achieved by cell fusion of individual transfectants4 August 2009 | European Journal of Biochemistry, Vol. 269, No. 13Functional Assay Screens17 December 2009Overexpression of Manganese Superoxide Dismutase Protects Lung Epithelial Cells against Oxidant InjuryAmerican Journal of Respiratory Cell and Molecular Biology, Vol. 24, No. 4Combinatorial gene expression using multiple episomal vectorsGene, Vol. 243, No. 1-2Enhanced neuronal differentiation of NTera-2 cells expressing neuronally restricted β2 adrenergic receptorBrain Research, Vol. 799, No. 2The sulphonylurea receptor SUR1 regulates ATP-sensitive mouse Kir6.2 K + channels linked to the green fluorescent protein in human embryonic kidney cells (HEK 293)22 September 2004 | The Journal of Physiology, Vol. 510, No. 2 Vol. 23, No. 3 Follow us on social media for the latest updates Metrics Downloaded 59 times History Published online 7 August 2018 Published in print September 1997 Information© 2018 Author(s)PDF download