Sonic hedgehog induces the differentiation of ventral neuronal cell types in explants derived from prospective forebrain regions of the neural plate. Neurons induced in explants derived from both diencephalic and telencephalic levels of the neural plate express the LIM homeodomain protein Isl-1, and these neurons possess distinct identities that match those of the ventral neurons generated in these two subdivisions of the forebrain in vivo. A single inducing molecule, SHH, therefore appears to mediate the induction of distinct ventral neuronal cell types along the entire rostrocaudal extent of the embryonic central nervous system (Ericson, 1995).

During differentiation of the embryonic anterior pituitary, distinct hormone cell types are generated in a precise temporal and spatial order from an apparently homogenous ectodermal primordium. The anterior pituitary derives from Rathke's pouch (RP), a specialized region of the oral roof ectoderm. The posterior pituitary derives from the infundibulum (INF) an evagination of the ventral diencephalon. Evidence is provided that in RP, the coordinate control of progenitor cell identity, proliferation and differentiation is imposed by spatial and temporal restrictions in FGF- and BMP-mediated signals. These signals derive from adjacent neural and mesenchymal signaling centers: the infundibulum and ventral juxtapituitary mesenchyme (VJM), respectively. The infundibulum appears to have a dual signaling function, serving initially as a source of BMP4 and subsequently of FGF8. The onset of FGF8 expression in the INF coincides with that of Lhx3 expression in RP. The ability of the INF over the period E10.5 to E12.5 to extinguish Isl1 (in the dorsal aspect of the RP) and promote Lhx3 in the same region corresponds more closely to the temporal expression of FGF8 than of BMP4. FGF8 can mimic the ability of the INF to repress Isl1 and maintain Lhx3 expression in explants. In vitro, FGFs promote the proliferation of progenitor cells, prevent their exit from the cell cycle and contribute to the specification of progenitor cell identity. Late FGF8 signaling controls corticotroph differentiation in the dorsal Lhx3+, Isl1- domain. Maintained FGF8 signaling from the INF expands still further the dorsal corticotroph prrogenitor population; as a consequence, the most ventral of these progenitors become located beyond range of FGF signaling. Since these progenitors are also beyond range of, or by this time refractory to, BMP2/7 signals (derived from the VJM), they progress to an ACTH+ definitive corticotroph state (Ericson, 1998).

In 2- to 7-day chick embryos, genes of the LIM homeobox family are expressed differentially among cranial motor nuclei. Whereas Islet-1 is expressed by motor neurons of all cranial nerves, Islet-2 is expressed only in nuclei that contain somatic motor neurons and transiently in specialized populations of contralateral vestibuloacoustic efferent neurons. Lim-3 is expressed in the hypoglossal and accessory abducens nuclei only; Lim-1 and Lim-2 are not expressed by cranial motor neurons (Varela-Echavarria, 1996).

A novel protein, nuclear LIM interactor (NLI) has been isolated, that specifically associates with single LIM domains in all nuclear LIM proteins tested. NLI is expressed in the nuclei of diverse neuronal cell types and is coexpressed with a target interactor Islet-1 during the initial stages of motor neuron differentiation, suggesting the mutual involvement of these proteins in the differentiation process. A number of NLI+/Isl1+ neurons also express NF-M, a neuronal phenotypic marker that is classically considered to be one of the earliest characteristics of nerve cells. NLI and ISl1 positive neurons seem to precede NF-M expression in at least some motor neurons. NLI is expressed shortly after neurons leave the cell cycle. Because ISl1 is required for motor neuron differentiation, the interaction with NLI throughout this process is likely to be of functional importance. The broad range of interactions betwen NLI and LIM-containing transcription factors suggests the utilization of a common mechanism to impart unique cell fate instructions (Jurata, 1996).

The ventral domain of RP serves as the origin of thyrotrophs, defined by expression of the alpha-glycoprotein and thyroid stimulating hormone beta subunits. The continued proliferation of progenitor cells in the dorsal domain of RP (stimulated by FGF8 signals derived from the INF) results in the progressive ventral displacement of thyrotroph progenitors such that they come to be located beyond the range of FGF8 signaling. The ventral juxtapituitary mesenchyme appears to serve as a later source of BMP2 and BMP7. BMPs have no apparent effect on cell proliferation but instead appear to act with FGFs to control the initial selection of thyrotroph and corticotroph progenitor identity. BMPs promote prospective thyrotroph differentiation and suppress corticotroph differentiation. BMPs expressed by the VJM promote Isl1 expression in the ventral domain of RP. Ultimately cells in the ventral domain begin to express TSHbeta and alpha-glycoprotein, having become established as definitive thyrotrophs (Ericson, 1998).

A complementary DNA (1392 bp) encoding a protein with high homology to rat reversion-induced LIM (RIL) protein was cloned from rat hepatocytes by differential screening of a subtractive (normoxic minus hypoxic) lambda GEM-2 cDNA library. This cDNA clone, denoted CLP-36, encodes a 327-amino-acid (aa) protein that contains a restrictively conserved LIM (a Cys-rich domain with consensus aa sequence C-X2-C-X17-19-H-X2-C-X2-C-X2-C-X16-20-C-X2-C/H/D). It shares an overall 45.1% identity to a rat LIM domain RIL protein, with 62.0% identity in the N terminus (aa 1-89) and 50.0% identity in the C terminus (aa 188-327). The rat CLP-36 mRNA is expressed most abundantly in heart, lung and liver, but only moderately in spleen and skeletal muscle. Analysis of total RNA extracted from normoxic or hypoxic rat hepatocytes, with a fragment of clone CLP-36 as probe, demonstrates a single band with a mobility corresponding to a size of 1.4 kb, whose level was significantly decreased during chemical hypoxia (Wang, H., 1995).

Many eukaryotic genes are regulated by cAMP through a conserved cAMP response element (CRE). In the pancreatic islet cell line Tu6, a well-characterized CRE in the somatostatin gene does not provide cAMP responsiveness but functions as an essential element for its basal activity. DNA-binding and functional analyses indicate that the cAMP-responsive factor CREB regulates somatostatin expression in these cells without requirement for phosphorylation at the protein kinase A-regulated Ser-133 phosphorylation site. In addition to the CRE site, cell-specific expression of the somatostatin gene requires a second promoter element, which binds the recently characterized LIM family protein Isl-1. Thus, Isl-1 and CREB appear to synergize on the somatostatin promoter to stimulate high-level expression in Tu6 cells. The ability of CREB to function in a phosphorylation-independent manner suggests a mechanism by which this protein can regulate gene transcription (Leonard, 1992).

Hearts of mice lacking Isl1, a LIM homeodomain transcription factor, are completely missing the outflow tract, right ventricle, and much of the atria. isl1 expression and lineage tracing of isl1-expressing progenitors demonstrates that Isl1 is a marker for a distinct population of undifferentiated cardiac progenitors that give rise to the cardiac segments missing in isl1 mutants. Isl1 function is required for these progenitors to contribute to the heart. In isl1 mutants, isl1-expressing progenitors are progressively reduced in number, and FGF and BMP growth factors are downregulated. These studies define two sets of cardiogenic precursors, one of which expresses and requires Isl1 and the other of which does not. The results have implications for the development of specific cardiac lineages, left-right asymmetry, cardiac evolution, and isolation of cardiac progenitor cells (Cai, 2003).

The cell types of the inner ear originate from the otic placode, a thickened layer of ectoderm adjacent to the developing hindbrain. The placode invaginates and forms the otic pit, which pinches off as a small vesicle called the otocyst. Presumptive cochleovestibular neurons delaminate from the anterior ventral part of the otocyst and form the cochleovestibular ganglion of the inner ear. The LIM/homeodomain protein islet-1 is expressed in cells of the ventral part of the otic placode and this ventral expression is maintained at the otic pit and the otocyst stages. Auditory and vestibular neurons originate from this islet-1-positive zone of the otocyst, and these neurons maintain islet-1 expression until adulthood. Islet-1 becomes up-regulated in the presumptive sensory epithelia of the inner ear in regions that are defined by the expression domains of BMP4. The up-regulation of islet-1 in developing inner ear hair and supporting cells is accompanied by down-regulation of Pax-2 in these cell types. Islet-1 expression in hair and supporting cells persists until early postnatal stages, when the transcriptional regulator is down-regulated in hair cells. These data are consistent with a role for islet-1 in differentiating inner ear neurons and sensory epithelia cells, perhaps in the specification of cellular subtypes in conjunction with other LIM/homeodomain proteins (Li, 2004).

The LIM domain homeobox gene isl-1 is a positive regulator of islet cell-specific proglucagon gene transcription. isl-1 is expressed in all 4 islet cell types, but a role for Isl-1 in the regulation of insulin gene expression has not been demonstrated, and the genetic targets for Isl-1 in the pancreas remain unknown. The proximal rat proglucagon gene promoter binds an amino-terminally truncated Trp-E-isl-1 fusion protein that lacks the LIM domains. The proglucagon gene promoter also binds full-length in vitro translated Isl-1 containing the intact LIM domains. Isl-1 antisera detects binding of proglucagon gene sequences to Isl-1 present in a slowly-migrating complex in nuclear extracts from InR1-G9 islet cells. These data demonstrate that the LIM domain homeobox gene isl-1 is not constrained from DNA binding by its LIM domains and that it functions as a positive regulator of proglucagon gene transcription in the endocrine pancreas (Wang, M., 1995).

The mammalian pancreas is a specialized derivative of the primitive gut endoderm and controls many homeostatic functions through the activity of its component exocrine acinar and endocrine islet cells. The LIM homeodomain protein ISL1 is expressed in all classes of islet cells in the adult ; its expression in the embryo is initiated soon after the islet cells have left the cell cycle. ISL1 is also expressed in mesenchymal cells that surround the dorsal but not ventral evagination of the gut endoderm, which together comprise the pancreatic anlagen. To define the role of ISL1 in the development of the pancreas, acinar and islet cell differentiation were analyzed in mice deficient in ISL1 function. Dorsal pancreatic mesenchyme does not form in ISL1-mutant embryos and there is an associated failure of exocrine cell differentiation in the dorsal but not the ventral pancreas. There is also a complete loss of differentiated islet cells. Exocrine, but not endocrine, cell differentiation in the dorsal pancreas can be rescued in vitro by provision of mesenchyme derived from wild-type embryos. These results indicate that ISL1, by virtue of its requirement for the formation of dorsal mesenchyme, is necessary for the development of the dorsal exocrine pancreas, and also that ISL1 function in pancreatic endodermal cells is required for the generation of all endocrine islet cells (Ahlgren, 1997).

The distal portion of the rat insulin I gene 5'-flanking DNA contains two sequence elements, the Far and FLAT elements, that can function in combination, but not separately, as a beta-cell-specific transcriptional enhancer. Several cDNAs encoding proteins that bind to the FLAT element have been isolated. Two of these cDNAs, cdx-3 and lmx-1, represent homeo box containing mRNAs with restricted patterns of expression. The protein encoded by lmx-1 also contains two amino-terminal cysteine/histidine-rich 'LIM' domains. Both cdx-3 and lmx-1 can activate transcription of a Far/FLAT-linked gene when expressed in a normally non-insulin-producing fibroblast cell line. Furthermore, in fibroblasts expressing transfected beta-cell lmx-1, the addition of the Far-binding, basic helix-loop-helix protein shPan-1 (the hamster equivalent of human E47) causes a dramatic synergistic activation. ShPan-1 causes no activation in fibroblasts expressing transfected cdx-3 or the related LIM-homeodomain protein isl-1. Deletion of one or both of the LIM domains from the 5' end of the lmx-1 cDNA removes this synergistic interaction with shPan-1 without any loss of basal transcriptional activation. It is concluded that beta-cell lmx-1 functions by binding to the FLAT element and interacting through the LIM-containing amino terminus with shPan-1 bound at the Far element. These proteins form the minimal components for a functional mini-enhancer complex (German, 2002).

The anatomical and functional organization of dorsal thalamus (dTh) and ventral thalamus (vTh), two major regions of the diencephalon, is characterized by their parcellation into distinct cell groups, or nuclei, that can be histologically defined in postnatal animals. However, because of the complexity of dTh and vTh and difficulties in histologically defining nuclei at early developmental stages, understanding of the mechanisms that control the parcellation of dTh and vTh and the differentiation of nuclei is limited. A set of regulatory genes, which include five LIM-homeodomain transcription factors (Isl1, Lhx1, Lhx2, Lhx5, and Lhx9) and three other genes (Gbx2, Ngn2, and Pax6), have been defined that are differentially expressed in dTh and vTh of early postnatal mice in distinct but overlapping patterns that mark nuclei or subsets of nuclei. These genes exhibit differential expression patterns in dTh and vTh as early as embryonic day 10.5, when neurogenesis begins; the expression of most of them is detected as progenitor cells exit the cell cycle. Soon thereafter, their expression patterns are very similar to those observed postnatally, indicating that unique combinations of these genes mark specific cell groups from the time they are generated to their later differentiation into nuclei. These findings suggest that these genes act in a combinatorial manner to control the specification of nuclei-specific properties of thalamic cells and the differentiation of nuclei within dTh and vTh. These genes may also influence the pathfinding and targeting of thalamocortical axons through both cell-autonomous and non-autonomous mechanisms (Nakagawa, 2001).

The dTh is parcellated into over one dozen nuclei. The principal sensory nuclei, dorsal lateral geniculate (dLG), ventroposterior (VP), and ventral medial geniculate (MGv), relay sensory information from the periphery to primary sensory areas of the neocortex, visual, somatosensory, and auditory, respectively, via thalamocortical axons (TCAs). Other nuclei, such as posterior (Po) and lateral posterior (LP), project broadly to cortex. The vTh has three major nuclei: reticular (RT), zona incerta (ZI), and ventral lateral geniculate (vLG). Different domains of embryonic vTh are required for TCA pathfinding (Nakagawa, 2001 and references therein).

The vTh and dTh have been defined as adjacent domains of the embryonic diencephalic alar plate based on expression of the homeodomain transcription factors Dlx2 and Gbx2, respectively, and restrictions in cell movement. However, little is known about the organization of embryonic dTh and vTh into discrete cell groups that presage their differentiation into nuclei, because the morphology and connections that define nuclei emerge late in development. The LIM-homeodomain (LIM-HD) family of transcription factors, as well as Gbx2, Pax6, and Neurogenin2, are candidates to be differentially expressed within dTh and vTh and control their parcellation. The LIM-HD genes Lhx1 and Lhx5 are expressed in early embryonic diencephalon, Lhx2 and Lhx9 in embryonic dTh, and Isl1 in adult RT. LIM-HD genes are intriguing because their unique combinations mark subsets of spinal neurons and specify their phenotypes, including axonal projections. Gbx2 is expressed broadly early in dTh and later in a subset of nuclei that require it for their differentiation, as well as for the development of the TCA projection. Pax6, a paired-box transcription factor, is expressed broadly early in vTh, later more discretely, and is required for development of RT, ZI, and vLG and TCA pathfinding. Ngn2, a basic helix-loop-helix transcription factor expressed in a subset of progenitor cells in dTh, is required for sensory neuron differentiation and dorsoventral patterning of the telencephalon. These regulatory genes are expressed in distinct yet often overlapping patterns, suggesting that they cooperate to control the specification and differentiation of thalamic nuclei and cell types (Nakagawa, 2001 and references therein).

The sets of genes that are expressed in dTh and vTh are distinct from one another and similar to those expressed in dorsal and ventral spinal cord, respectively. This similarity suggests that the expression patterns in thalamus might be established by mechanisms similar to those in spinal cord. In spinal cord, inductive signals from the roof plate and floor plate control neuronal fate along the dorsoventral axis. Signals from the roof plate, such as TGFß family members, are required in dorsal spinal cord for the induction of Lhx2 and Lhx9, which define D1A and D1B interneurons, respectively. In ventral spinal cord, distinct classes of motor neurons and ventral interneurons are generated by a graded signaling activity of Shh. Shh controls these neural fates by establishing different progenitor cell populations defined by their expression of Pax6 and Nkx2.2. Pax6 establishes distinct populations of ventral progenitor cells and controls the identity of motor neurons and V1 and V2 interneurons, whereas Nkx2.2 specifies the identity of V3 interneurons at a more ventral location. These genes appear to be essential intermediaries for Shh to regulate the differential expression of LIM-HD proteins, including Lhx1, Lhx3, Lhx4, Lhx5, Isl1, and Isl2. In diencephalon, Shh is transiently expressed as early as E9.5 in the zona limitans intrathalamica , which at this stage is a narrow cell domain interposed between prospective dTh and vTh. Similar to ventral spinal cord, Nkx2.2 and Pax6 are also expressed in progenitor cells in vTh. Shh induces in vitro the expression of Isl1 in chick forebrain explants and neuroepithelial cells from rat forebrain. Therefore, ZLI-derived Shh may specify progenitor cell types in vTh to produce different neuronal subtypes, which are determined by the subset of LIM-HD and other transcription factors expressed by these neurons. Interestingly, dTh, which is adjacent to the ZLI, does not express any of the LIM-HD genes induced by Shh and expressed in vTh. Ngn2, which is expressed by progenitor cells of dTh but not vTh, could act to limit the responsiveness of dTh to an Shh-mediated induction of vTh-type LIM-HD genes, which may be a crucial step in regionalization of the diencephalon (Nakagawa, 2001 and references therein).

It is believed that mouse dentition is determined by a prepatterning of the oral epithelium into molar and incisor regions. The LIM homeodomain protein Islet1 (ISL1) is involved in the regulation of differentiation of many cell types and organs. During odontogenesis, Islet1 is found to be exclusively expressed in epithelial cells of the developing incisors but not during molar development. Early expression of Islet1 in presumptive incisor epithelium is coincident with expression of Bmp4, which acts to induce Msx1 expression in the underlying mesenchyme. To define the role of ISL1 in the acquisition of incisor shape, regulation of Islet1 expression in mandibular explants has been analyzed. Local application of bone morphogenetic protein 4 (BMP4) in the epithelium of molar territories (proximal) stimulates Islet1 expression and has been carried out either by bead implantation or by electroporation. Inhibition of BMP signalling with Noggin results in a loss of Islet1 expression. Inhibition of Islet1 in distal epithelium (producing incisors) results in a loss of Bmp4 expression and a corresponding loss of Msx1 expression, indicating that a positive regulatory loop exists between ISL1 and BMP4 in distal epithelium. Ectopic expression of Islet1 in proximal epithelium produces a loss of Barx1 expression in the mesenchyme and results in inhibition of molar tooth development. Using epithelial/mesenchymal recombinations it has been shown that at E10.5 Islet1 expression is independent of the underlying mesenchyme whereas at E12.5 when tooth shape specification has passed to the mesenchyme, Islet1 expression requires distal (presumptive incisor) mesenchyme. Islet1 thus plays an important role in regulating distal gene expression during jaw and tooth development (Mitsiadis, 2003).