Lim1
This paper reports on cloning, sequence analysis, and developmental expression pattern of lim1, a member of the LIM class homeobox gene family in the mouse. lim1 cDNA encodes a predicted 406 amino acid protein that is 93% identical with the product of the Xenopus LIM class homeobox gene Xlim1. lim1 expression from day 8.5 post coitum onward has been characterized. Northern blot analysis of RNA transcripts indicate that lim1 is expressed both during embryogenesis and in the adult brain. Analysis by whole-mount and section in situ hybridization shows lim1 expression in the central nervous system from the telencephalon through the spinal cord and in the developing excretory system including the pronephric region, mesonephros, nephric duct, and metanephros. In the metanephros, lim1 is strongly expressed in renal vesicles and S-shaped bodies, and transcripts are also detected in the ureteric branches (Fujii, 1999).
The sequence and genomic organization of the mouse Lim1 gene have been determined. The mouse Lim1 gene has five coding exons. The Lim1 transcription initiation start site was determined by 5' RACE. Results indicate that the first exon encodes the translation initiation codon and a 1360-bp 5' untranslated region. Sequence analysis of the 450-bp upstream of the transcription start site reveals the presence of a CATTAA motif at -32 bp and a CAATT box located in reverse orientation at -68 bp. HNF3 beta and Pbx1 binding sites have also been identified. Like most LIM domain encoding genes, the LIM domains of Lim1 are each encoded on separate and adjacent exons. Knowledge of the sequence and structure of the mouse Lim1 gene provides important information for the genetic manipulation of the Lim1 locus (Li, 1999).
To investigate Lim1 function during gastrulation, transcript depletion through DEED antisense oligonucleotides was used in Xenopus and cell transplantation was used in mice. Xenopus embryos depleted of Lim1 lack anterior head structures and fail to form a proper axis as a result of a failure of gastrulation movements, even though mesodermal cell identities are specified. Similar disruption of cell movements in the mesoderm is also observed in Lim1-/- mice. Paraxial protocadherin (PAPC) expression is lost in the nascent mesoderm of Lim1-/- mouse embryos and in the organizer of Lim1-depleted Xenopus embryos; the latter can be rescued to a considerable extent by supplying PAPC exogenously. It is concluded that a primary function of Lim1 in the early embryo is to enable proper cell movements during gastrulation (Hukriede, 2003).
Lim1 is a homeobox gene expressed in the organizer region of mouse embryos. To investigate the role of Lim1 during embryogenesis, a targeted deletion of the Lim1 gene was generated in embryonic stem cells. Embryos homozygous for the null allele lack anterior head structures but the remaining body axis develops normally. A partial secondary axis develops anteriorly in some mutant embryos. Lim1 is thus an essential regulator of the vertebrate head organizer (Shawlot, 1995).
Recent experiments have implicated the visceral endoderm in anterior neural induction in mouse. The visceral endoderm is an extraembryonic tissue that surrounds the epiblast of the egg cylinder stage embryo. During gastrulation the visceral endoderm is replaced by definitive endoderm that derives from the anterior portion of the primitive streak. Although no morphological asymmetries are apparent in the visceral endoderm, molecular studies have shown that a distinct anterior-posterior pattern exists in the visceral endoderm prior to the formation of the primitive streak. These studies reveal that the VE-1 antigen and the homeobox genes Hesx1/Rpx and Hex are expressed in the anterior visceral endoderm that underlies the ectoderm fated to form the anterior portion of the neural plate. Ablation experiments have shown that, if the anterior visceral endoderm is removed at the early streak stage, expression of Hesx1/Rpx in the anterior neuroectoderm in late streak/headfold stage embryos is absent or greatly reduced (Shawlot, 1999 and references therein).
Lim1 is a homeobox gene expressed in the extraembryonic anterior visceral endoderm and in primitive streak-derived tissues of early mouse embryos. Mice homozygous for a targeted mutation of Lim1 lack head structures anterior to rhombomere 3 in the hindbrain. To determine in which tissues Lim1 is required for head formation and its mode of action, chimeric mouse embryos were generated and tissue layer recombination explant assays were performed. In chimeric embryos in which the visceral endoderm is composed of predominantly wild-type cells, Lim1 -/- cells are able to contribute to the anterior mesendoderm of embryonic day 7.5 chimeric embryos but embryonic day 9.5 chimeric embryos display a range of head defects. In addition, early somite stage chimeras generated by injecting Lim1 -/- embryonic stem cells into wild-type tetraploid blastocysts lack forebrain and midbrain neural tissue. Furthermore, in explant recombination assays, anterior mesendoderm from Lim1 -/- embryos is unable to maintain the expression of the anterior neural marker gene Otx2 in wild-type ectoderm. In complementary experiments, embryonic day 9.5 chimeric embryos in which the visceral endoderm is composed of predominantly Lim1 -/- cells and the embryo proper of largely wild-type cells, also phenocopies the Lim1 -/- headless phenotype. These results indicate that Lim1 is required in both primitive streak-derived tissues and visceral endoderm for head formation and that its inactivation in these tissues produces cell non-autonomous defects. A double assurance model in which Lim1 regulates sequential signaling events required for head formation in the mouse is discussed (Shawlot, 1999).
Recent embryological and genetic experiments have suggested that the anterior visceral endoderm and the anterior primitive streak of the early mouse gastrula function as head- and trunk-organizing centers, respectively. HNF3beta and Lim1 are coexpressed in both organizing centers suggesting synergistic roles for these genes in regulating organizer functions and hence axis development in the mouse embryo. To investigate this possibility, compound HNF3beta and Lim1 mutant embryos were generated. An enlarged primitive streak and a lack of axis formation were observed in double mutant but not in single homozygous mutant embryos. Chimera experiments indicate that the primary defect in these double homozygous mutants is due to loss of activity of HNF3beta and Lim1 in the visceral endoderm. Altogether, these data provide evidence that these genes function synergistically to regulate organizer activity of the anterior visceral endoderm. Moreover, double mutant embryos also exhibit defects in mesoderm patterning that are likely due to lack of specification of anterior primitive streak cells (Perea-Gomez, 1999).
The first morphological sign of A-P pattern in the epiblast of the mouse embryo is the site of formation of the primitive streak at the posterior end of the embryo. The genetic pathway that initiates primitive streak formation remains to be elucidated, but expression of T on one side of the epiblast at the onset of gastrulation marks posterior primitive streak cells. In HNF3beta,Lim1 double mutant embryos, T expression in the epiblast is no longer restricted posteriorly, but is instead expressed throughout the epiblast by the mid-streak stage. Thus, A-P polarity of the epiblast is abnormal in HNF3beta,Lim1 embryos and widespread expression of T strongly suggests that mutant epiblast cells are transformed into primitive streak cells. The loss of epiblast cells is confirmed by the absence and reduction of expression of Otx2 and Oct4, respectively. In addition, mid-streak-stage embryos also show ectopic mesoderm formation as demonstrated by the expression of MesP1 and Lefty2. As a consequence of these early patterning defects, ectoderm and neurectoderm cells that are derived from distal and anterior epiblast cells are missing in these embryos at 7.5-7.75 d.p.c. These epiblast defects are not observed in single homozygous HNF3beta and Lim1 mutants. Altogether, these data demonstrate that HNF3beta and Lim1 function synergistically to establish A-P patterning of the epiblast and to restrict primitive streak formation to the posterior side of mouse embryos (Perea-Gomez, 1999).
Investigation of the developmental fates of cells in the endodermal layer of the early bud stage mouse embryo has revealed a regionalized pattern of distribution of the progenitor cells of the yolk sac endoderm and the embryonic gut. By tracing the site of origin of cells that are allocated to specific regions of the embryonic gut, it was found that by late gastrulation, the respective endodermal progenitors are already spatially organized in anticipation of the prospective mediolateral and anteriorposterior destinations. The fate-mapping data further showed that the endoderm in the embryonic compartment of the early bud stage gastrula still contains cells that will colonize the anterior and lateral parts of the extraembryonic yolk sac. In the Lhx1(Lim1)-null mutant embryo, the progenitors of the embryonic gut are confined to the posterior part of the endoderm. In particular, the prospective anterior endoderm is sequestered to a much smaller distal domain, suggesting that there may be fewer progenitor cells for the anterior gut that is poorly formed in the mutant embryo. The deficiency of gut endoderm is not caused by any restriction in endodermal potency of the mutant epiblast cells but more likely the inadequate allocation of the definitive endoderm. The inefficient movement of the anterior endoderm, and the abnormal differentiation highlighted by the lack of Sox17 and Foxa2 expression, may underpin the malformation of the head of Lhx1 mutant embryos (Tam, 2004).
Numerous studies have identified the roof plate as an embryonic signaling center critical for dorsal central nervous system patterning, but little is known about mechanisms that control its formation and its separation from clonally related neural crest cells and dI1 sensory interneurons. The LIM homeodomain transcription factor, Lmx1a, mutated in the dreher mouse, acts to withdraw dorsal spinal cord progenitors from the cell cycle and simultaneously direct their differentiation into functional roof plate cells. Lmx1a cell-autonomously represses the dI1 progenitor fate, distinguishing the roof plate and dI1 interneuron programs, two major developmental programs of the dorsal neural tube. Lmx1a is not directly involved in neural crest development. Bmp signaling from epidermal ectoderm is necessary and sufficient for inducing Lmx1a and other co-factors that also regulate the extent of roof plate induction. It is concluded that Lmx1a controls multiple aspects of dorsal midline patterning and is a major mediator of early Bmp signaling in the developing spinal cord (Chizhikov, 2004).
Motor neurons located at different positions in the embryonic spinal cord innervate distinct targets in the periphery, establishing a topographic neural map. The topographic organization of motor projections depends on the generation of subclasses of motor neurons that select specific paths to their targets. A family of LIM homeobox genes has been cloned in the chick. The combinatorial expression of four of these genes, Islet-1, Islet-2 (homologs of Drosophila Islet), Lim-1, and Lim-3, defines subclasses of motor neurons that segregate into columns in the spinal cord and select distinct axonal pathways. Thus the combination of LIM domain proteins serve to code motor neuron identity in the spinal cord (Tsuchida, 1994).
These LIM homeodomain proteins are expressed prior to the formation of distinct motor axon pathways and before motor columns appear. Depending on their arrangement in columns and eventual synaptic targets, motor neurons of the chick brain stem are designated as belonging to somatic motor (sm) visceral motor (vm), or branchiomotor (bm) classes or to the ipsilateral or contralateral vestibuloacoustic effect neuronal population. Sm neurons innervate muscle derived from the paraxial mesoderm and prechordal plate mesoderm. Bm, vm and vestibuloacoustic axons extend dorsolaterally for some distance through the neuroepithelium before converging on large single exit points within the dorsal neural tube (alar plate). Bm neurons innervate muscle derived from paraxial mesoderm within the branchial arches, while vm neurons innervate parasympathetic ganglia associated with lacrimal and salivary glands or neuronal plexuses that innervate smooth muscle; vestibuloacoustic efferent neurons innervate the hair cells of the inner ear. Subpopulations of spinal motor neurons within specific locations in the spinal cord and distinct targets in the periphery express different combinations of LIM homeobox genes. Sm neurons of the medial division of the median motor column express Islet-1, Isl-2, and Lim-3, while those of the lateral division of the median motor column and the medial division of the later motor column express Isl-1 and Isl-2. Sm neurons of the lateral division of the lateral motor column express Lim-1 and Isl-2. Since the lateral motor column is present only at limb levels, Lim-1 expression is restricted to these levels of the neuraxis. At early stages, visceral motor neurons express both Isl-1 and Isl-2, but after their migration to form the column of Terni, only a subset of these neurons continues to express Isl-1. These genes are good candidates to confer target specificity upon motor neuron classes, since they are expressed at times before the motor columns have fully segregated and before axons have reached their targets (Tsuchida, 1994).
The generation of distinct classes of motor neurons is an early step in the control of vertebrate motor behavior. To study the interactions that control the generation of motor neuron subclasses in the developing avian spinal cord, in vivo grafting studies were performed in which either the neural tube or flanking mesoderm was displaced between thoracic and brachial levels. The positional identity of neural tube cells and motor neuron (MN) subtype identity was assessed by Hox and LIM homeodomain protein expression. Brachial (B) levels of the median motor column (MMC) are organized into three columns: neurons of the medial MMC (MMCM) co-express Isl1, Isl2 and Lim3; neurons of the medial lateral motor column (LMCM) co-express Isl1 and Isl2, and motoneurons of the lateral LMC (LMCL) coexpress Isl2 and Lim1. At thoracic (T) levels motoneurons are also organized into three columns: MMCM neurons; lateral MMC neurons that coexpress Isl1 and Isl2 but not Lim3, and dorsomedially positioned Column of Terni (CT) neurons that express only Isl1. Grafts of 13-15 segment quail T neural tube were placed rostrally at the B level of 12-15 segment chick hosts. Marker and morphological analysis reveals that grafted neural cells divert their normal T fates and their neuronal progeny acquire the molecular properties of B MNs. These changes in the neural tube are restricted to a limited time frame. The rostrocaudal identity of neural cells is plastic at the time of neural tube closure and is sensitive to positionally restricted signals from the paraxial mesoderm. Such paraxial mesodermal signals appear to control the rostrocaudal identity of neural tube cells and the columnar subtype identity of motor neurons. Analysis of neural Hoxc8 expression provides evidence that the change in cell identity after neural tube displacement is not restricted to the MNs; the change occurs in a graded manner along the rostrocaudal axis of the spinal cord, and is associated with both a rostral and caudal respecification in cell fate. In contrast, neural tube grafts between B and T levels do not change the pattern of Hoxc8 expression in the flanking paraxial mesodem. These results suggest that the generation of motor neuron subtypes in the developing spinal cord involves the integration of distinct rostrocaudal and dorsoventral patterning signals that derive, respectively, from paraxial and axial mesodermal cell groups (Ensini, 1998).
The diversification of neuronal cell types in the vertebrate central nervous system depends on inductive signals provided by local organizing cell groups of both neural and nonneural origin. The link between neuronal birth date, migratory pattern, and identity is also evident in the generation of motor neurons in the spinal cord. These conserved features are particularly apparent for motor neurons of the lateral motor column (LMC). This class of motor neurons is generated selectively at brachial and lumbar levels of the spinal cord, and their axons innervate target muscles in the limb. Within the LMC, motor neurons can be further divided into two subclasses: medial LMC neurons that project to ventrally derived limb muscles, and lateral LMC neurons that project to dorsally derived limb muscles. Motor neurons destined to form the medial LMC leave the cell cycle before lateral LMC neurons; as a consequence, prospective lateral LMC neurons emerge from the ventricular zone and migrate past medial LMC neurons to their final position. The time of generation and the distinct migratory environment represent two prominent differences between the development of lateral LMC neurons and other motor neurons. In addition, the total number of motor neurons generated at limb levels of the spinal cord is greater than that at nonlimb levels, presumably to accommodate the formation of the LMC (Sockanathan, 1998 and references).
All somatic motor neurons initially express Isl1 and Isl2, and most maintain the expression of these genes. Lateral LMC neurons, however, extinguish Isl1 and initiate Lim1 expression as they begin to migrate past medial LMC neurons, thus acquiring a unique LIM homeobox gene code. Studies of LIM homeobox gene function in vertebrates and invertebrates have provided evidence that this gene family has a role in motor neuron differentiation and axon pathfinding. The diversification of motor neuron subtypes is initiated by inductive signals from the axial and paraxial mesoderm that operate along the dorsoventral and rostrocaudal axes of the neural tube. However, medial and lateral LMC motor neurons are generated from progenitor cells that occupy the same dorsoventral and rostrocaudal positions, and thus it is unlikely that mesodermal signals impose this distinction. The late birth date of lateral LMC neurons and their migration past early-born LMC neurons prompted a consideration of whether the fate of lateral LMC neurons might be directed by signals provided by early-born LMC neurons. This hypothesis invokes the idea that LMC motor neurons generated at early stages express a local but non-cell-autonomous signal that induces the lateral LMC phenotype in late-born LMC neurons. A retinoid-mediated signal provided by one subset of early-born spinal motor neurons (the medial) imposes a local variation in the number of motor neurons generated at different axial levels and also specifies the identity of a later-born subset of motor neurons (the lateral). Thus, in the vertebrate central nervous system the distinct fates of late-born neurons may be acquired in response to signals provided by early-born neurons (Sockanathan, 1998).
The mesencephalic and metencephalic region (MMR) of the vertebrate central nervous system develops in response to signals produced by the isthmic organizer (IsO). The LIM homeobox transcription factor Lmx1b is expressed within the chick IsO, where it is sufficient to maintain expression of the secreted factor wnt1. This paper shows that zebrafish express two Lmx1b orthologs, lmx1b.1 and lmx1b.2, in the rostral IsO; these genes are necessary for key aspects of MMR development. Simultaneous knockdown of Lmx1b.1 and Lmx1b.2 using morpholino antisense oligos results in a loss of wnt1, wnt3a, wnt10b, pax8 and fgf8 expression at the IsO, leading ultimately to programmed cell death and the loss of the isthmic constriction and cerebellum. Single morpholino knockdown of either Lmx1b.1 or Lmx1b.2 has no discernible effect on MMR development. Maintenance of lmx1b.1 and lmx1b.2 expression at the isthmus requires the function of no isthmus/pax2.1, as well as Fgf signaling. Transient misexpression of Lmx1b.1 or Lmx1b.2 during early MMR development induces ectopic wnt1 and fgf8 expression in the MMR, as well as throughout much of the embryo. It is proposed that Lmx1b.1- and Lmx1b.2-mediated regulation of wnt1, wnt3a, wnt10b, pax8 and fgf8 maintains cell survival in the isthmocerebellar region (O'Hara, 2005).
To begin to define the contribution of retinoid signaling to motor neuron differentiation, the pattern of expression of retinaldehyde dehydrogenase 2 (RALDH2) in the developing spinal cord was examined. At brachial levels, RALDH2 expression is first detected at stage 19, and at this and subsequent stages, expression in the ventral spinal cord appears to be restricted to motor neurons. By stage 27, when the medial motor column (MMC) and LMC have segregated, expression of RALDH2 is restricted to LMC neurons. Within the LMC, RALDH2 is expressed by both medial and lateral LMC neurons. A similar LMC-specific pattern of RALDH2 expression is detected at lumbar levels. Consistent with the restriction of RALDH2 expression to LMC neurons, no expression of the gene is detected in motor neurons at thoracic levels. The expression of RALDH2 in motor neurons at brachial and lumbar levels persists until at least stage 35, although from stage 29 onward, expression gradually becomes restricted to specific motor neuron pools. The only other site of RALDH2 expression in the spinal cord is in the roof plate, both at limb and nonlimb levels. These selective results show that (1) RALDH2 expression is initiated during the early phase of motor neuron generation at brachial levels of the spinal cord; (2) RALDH2 expression distinguishes developing LMC neurons from other somatic or visceral motor neurons, and (3) RALDH2 expression precedes the appearance of lateral LMC neurons marked by Isl2 and Lim1 expression (Sockanathan, 1998).
The number of Isl+ motor neurons was counted in brachial ventral/floor plate (vf) explants grown either alone or with retinol (Rol), a metabolic precursor of retinoic acid, or with all-trans retinoic acid (RA). The number of Isl+ motor neurons in [vf] explants grown in the presence of either Rol or RA is increased by 60%. The detection of an increase in motor neuron number with Rol, as well as with RA, indicates that explants grown in medium with no added retinoid are deprived of the metabolic substrate required for synthesis of RA by RALDH2. To examine further the involvement of RALDH2 activity in the control of motor neuron number, thoracic [vf] explants, which do not express RALDH2, were exposed to Rol or RA and the number of Isl+ motor neurons measured. In contrast to results obtained with brachial level explants, exposure of thoracic [vf] explants to Rol does not increase motor neuron number, whereas RA similarly induces a 60% increase in Isl+ motor neurons. Taken together, these results provide evidence that (1) retinoids increase the number of motor neurons; (2) the increase in motor neuron number detected after exposure of brachial [vf] explants to Rol is correlated with the synthesis of active retinoids by RALDH2 activity, and (3) the apparent requirement for RALDH2-generated retinoids can be overcome by exogenous RA. The retinoid-induced increase in motor neuron number at brachial levels appears to result from an increase in the number of progenitor cells. These experiments suggest that, at limb levels, a RALDH2-generated LMC source of retinoids acts non-cell-autonomously to increase the number of motor neuron progenitors and, consequently, postmitotic motor neurons. Studies using an RAR antagonist show that retinoid receptor activation is required for the generation of lateral LMC neurons and for the control of motor neuron number. Maintenance of the lateral LMC phenotype appears to require ongoing retinoid signaling over the period that these neurons are migrating to their lateral position (Sockanathan, 1998).
RALDH2-dependent induction of lateral LMC neurons requires non-autonomous RA signaling. The onset of RALDH2 and Lim1 expression by lateral LMC neurons was examined. At stage 23, many Isl2+, Lim1+ lateral LMC neurons are still located medial to Isl1+, Isl2+ medial LMC neurons. These Isl2+, Lim1+ neurons do not express RALDH2, suggesting that their lateral LMC phenotype has not been acquired through cell-intrinsic RALDH2 activity. Many of the motor neurons that are located in an even more medial position, distant from RALDH2+ neurons, will populate the lateral LMC, but at this stage these neurons express Isl1/2 but not Lim1. These observations support the idea that the lateral phenotype of LMC neurons is acquired by virtue of the proximity of the neurons to a RALDH2-dependent signal provided by earlier-born LMC neurons. The late birth date of lateral LMC neurons requires that they migrate past early-born neurons to reach their final position. What role might this inside-out program of neuronal migration have in the establishment of the lateral LMC phenotype? The detection of late-born Isl2+, Lim1+ lateral LMC neurons in positions adjacent but medial to early-born RALDH2+ medial LMC neurons provides evidence that proximity to early-born neurons is sufficient to achieve a lateral LMC identity. The failure of late-born LMC neurons to migrate past medial LMC neurons might, however, have the consequence that some LMC neurons fail to be exposed to retinoid signals before they lose competence to respond. In this view, the migration of prospective lateral LMC neurons through early-born LMC neurons would achieve a rapid intermixing of inductive and responsive neurons and ensure that the entire population of late born LMC neurons efficiently encounters a local source of retinoid signals (Sockanathan, 1998).
LIM domains mediate protein-protein interactions. Within LIM-homeodomain proteins, the LIM domains act as negative regulators of the transcriptional activation function of the protein. The recently described protein Ldb1 (also known as NLI, or LIM domain-binding protein) binds LIM domains in vitro and synergizes with the LIM-homeodomain protein Xlim-1 in frog embryo microinjection experiments. The transcriptional activation domain of Xlim-1 has been localized to its carboxyl-terminal region; the interactions of the amino-terminally located LIM domains with Ldb1 have been characterized. Ldb1 binds LIM domains through its carboxyl-terminal region, and can form homodimers via its amino-terminal region. Optimal binding to Ldb1 requires tandem LIM domains, while single domains can bind with lower, yet clearly measurable efficiencies. In animal explant experiments, synergism of Ldb1 with Xlim-1 in the activation of downstream genes requires both the region containing the dimerization domain of Ldb1 and the region containing the LIM-binding domain. The role of Ldb1 may be to recruit other transcriptional activators depending on the promoter context and LIM-homeodomain partner involved (Breen, 1998).
The transcriptional activity of LIM-homeodomain (LIM-HD) proteins is regulated by their interactions with various factors that bind to the LIM domain. Reduced expression of single-stranded DNA-binding protein 1 (Ssdp1), which encodes a co-factor of LIM domain interacting protein 1 (Ldb1), in the mouse mutant headshrinker (hsk) disrupts anterior head development by partially mimicking Lim1 mutants. Although the anterior visceral endoderm and the anterior definitive endoderm, which together comprise the head organizer, are able to form normally in Ssdp1hsk/hsk mutants, development of the prechordal plate was compromised. Head development is partially initiated in Ssdp1hsk/hsk mutants, but neuroectoderm tissue anterior to the midbrain-hindbrain boundary is lost, without a concomitant increase in apoptosis. Cell proliferation is globally reduced in Ssdp1hsk/hsk mutants, and approximately half also exhibit smaller body size, similar to the phenotype observed in Lim1 and Ldb1 mutants. Ssdp1 contains an activation domain and is able to enhance transcriptional activation through a Lim1-Ldb1 complex in transfected cells, and Ssdp1 interacts genetically with Lim1 and Ldb1 in both head development and body growth. These results suggest that Ssdp1 regulates the development of late head organizer tissues and body growth by functioning as an essential activator component of a Lim1 complex through interaction with Ldb1 (Nishioka, 2005).
Ssdp1 mutants exhibit a global reduction in cell proliferation after E8.5 and an increase in apoptosis in somites at E9.0. These changes may be at the root of the abnormalities such as growth retardation and kinked neural tube that were observed in Ssdp1 mutants. Although the mechanism by which Ssdp1 regulates cell proliferation is unknown at present, growth retardation of Ssdp1+/hsk;Lim1+/- and Ssdp1+/hsk;Ldb1+/- compound mutants suggests involvement of a Ssdp1-Lim1-Ldb1 complex in this process. A shortened body axis was also observed in embryos lacking either Ldb1 or Lim1, supporting this hypothesis. However, if the Lim1 complex plays a major role in the regulation of cell proliferation and cell death, it must be through an indirect mechanism, since Lim1 is not expressed in all of the affected cells. It is conceivable that defective gastrulation movements or the inability of cells with reduced Lim1 complex activity to induce lateral plate mesoderm genes secondarily affects the proliferation and survival of surrounding cells. Furthermore, it is possible that Ssdp1 may also function independently of Lim1, in which case the Ldb1-Ssdp1 complex may regulate cell proliferation in a cell-autonomous manner by controlling the activities of transcription factors involved in cell cycle regulation and cell survival. Alternatively, Ssdp1 might play a direct role in the DNA replication process as a single stranded DNA-binding protein (Nishioka, 2005).
Analysis of hsk mutants shows that disruption of the Ssdp1 gene and the resulting reduction in Ssdp1 expression causes defects in the prechordal plate development and anterior truncations, with some mutants also exhibiting smaller body size. In vitro data have demonstrated that Ssdp1 acts as a coactivator that enhances transcriptional activation by the Lim1-Ldb1 complex. Moreover, genetic interactions between Ssdp1 and Lim1 or Ldb1 suggest that the phenotypes observed in Ssdp1 mutants very probably reflect reduced activity of a Lim1 complex. Together, these data demonstrate that Ssdp1 acts as an essential activator component of a Ssdp1-Lim1-Ldb1 complex in the development of the prechordal plate and body growth (Nishioka, 2005).
Lim1 encodes a LIM-class homeodomain transcription factor that is essential for head and kidney development. In the developing urogenital system, Lim1 expression has been documented in the Wolffian (mesonephric) duct, the mesonephros, metanephros and fetal gonads. Using a Lim1 lacZ knock-in allele in mice. a previously unreported urogenital tissue for Lim1 expression was identified, the epithelium of the developing Müllerian duct that gives rise to the oviduct, uterus and upper region of the vagina of the female reproductive tract. Lim1 expression in the Müllerian duct is dynamic, corresponding to its formation and differentiation in females and regression in males. Although female Lim1-null neonates have ovaries they lack a uterus and oviducts. A novel female mouse chimera assay was developed and revealed that Lim1 is required cell autonomously for Müllerian duct epithelium formation. These studies demonstrate an essential role for Lim1 in female reproductive tract development (Kobayashi, 2003).
To bypass the essential gastrulation function of Fgf8 and study its role in lineages of the primitive streak, a new mouse line, T-Cre, was used to generate mouse embryos with pan-mesodermal loss of Fgf8 expression. Surprisingly, despite previous models in which Fgf8 has been assigned a pivotal role in segmentation/somite differentiation, Fgf8 is not required for these processes. However, mutant neonates display severe renal hypoplasia with deficient nephron formation. In mutant kidneys, aberrant cell death occurs within the metanephric mesenchyme (MM), particularly in the cortical nephrogenic zone, which provides the progenitors for recurring rounds of nephron formation. Prior to mutant morphological changes, Wnt4 and Lim1 expression, which is essential for nephrogenesis, is absent in MM. Furthermore, comparative analysis of Wnt4-null homozygotes reveals concomitant downregulation of Lim1 and diminished tubule formation. These data support a model whereby FGF8 and WNT4 function in concert to induce the expression of Lim1 for MM survival and tubulogenesis (Berantoni, 2005).
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