Antenatal dexamethasone exposure differentially affects distinct cortical neural progenitor cells and triggers long-term changes in murine cerebral architecture and behavior.

Antenatal administration of synthetic glucocorticoids (sGC) is the standard of care for women at risk for preterm labor before 34 gestational weeks. Despite their widespread use, the type of sGC used and their dose or the dosing regimens are not standardized in the United States of America or worldwide. Several studies have identified neural deficits and the increased risk for cognitive and psychiatric disease later in life for children administered sGC prenatally. However, the precise molecular and cellular targets of GC action in the developing brain remain largely undefined. In this study, we demonstrate that a single dose of glucocorticoid during mid-gestation in mice leads to enhanced proliferation in select cerebral cortical neural stem/progenitor cell populations. These alterations are mediated by dose-dependent changes in the expression of cell cycle inhibitors and in genes that promote cell cycle re-entry. This leads to changes in neuronal number and density in the cerebral cortex at birth, coupled to long-term alterations in neurite complexity in the prefrontal cortex and hippocampus in adolescents, and changes in anxiety and depressive-like behaviors in adults.

Loss of the Sall3 gene leads to palate deficiency, abnormalities in cranial nerves, and perinatal lethality.

Members of the Spalt gene family encode putative transcription factors characterized by seven to nine C2H2 zinc finger motifs. Four genes have been identified in mice--Spalt1 to Spalt4 (Sall1 to Sall4). Spalt homologues are widely expressed in neural and mesodermal tissues during early embryogenesis. Sall3 is normally expressed in mice from embryonic day 7 (E7) in the neural ectoderm and primitive streak and subsequently in the brain, peripheral nerves, spinal cord, limb buds, palate, heart, and otic vesicles. We have generated a targeted disruption of Sall3 in mice. Homozygous mutant animals die on the first postnatal day and fail to feed. Examination of the oral structures of these animals revealed that abnormalities were present in the palate and epiglottis from E16.5. In E10.5 embryos, deficiencies in cranial nerves that normally innervate oral structures, particularly the glossopharyngeal nerve (IX), were observed. These studies indicate that Sall3 is required for the development of nerves that are derived from the hindbrain and for the formation of adjacent branchial arch derivatives.

Expression of the transcription factor, tailless, is required for formation of superficial cortical layers.

The gene tailless (tlx) encodes a forebrain-restricted transcription factor that is robustly expressed in progenitor cells of the ventricular and subventricular zones during neurogenesis. To investigate the role of tlx in neocortical development we generated a targeted deletion of tlx by homologous recombination. Here we compared the lamination, connectivity and patterning of cortical regions in adult tlx-/- mice and their wild-type littermates. We found first that neocortical thickness is reduced by 20% in mutant animals; most of this reduction is due to a diminution of supragranular layers, while layer I and layers IV through VI are relatively intact cytoarchitecturally. Consistent with this, the cross-sectional area of the corpus callosum is reduced by over 40%. Second, thalamocortical and intrinsic excitatory circuits in tlx-/- mice exhibit an essentially normal distribution from layer IV to the white matter, but are reduced superficial to layer IV. Finally, within parietal cortex of mutant mice a vibrissa-like pattern of cortical barrels is present in the expected rostro-caudal location. These observations indicate that loss of tlx function most severely affects generation and differentiation of neurons destined for superficial cortical layers. Thus, tlx may be important in sustaining the progenitor cell population throughout late prenatal development. Establishment of functional cortical areas, and development of basic patterns of thalamocortical and intra-cortical circuits occurs independently of tlx function.

A new member of the spalt like zinc finger protein family, Msal-3, is expressed in the CNS and sites of epithelial/mesenchymal interaction.

We have identified a new member of the spalt-like gene family in mice, msal-3. We compared the expression patterns of msal-3 and msal-1 during development and show that they exhibit overlapping yet exclusive patterns of expression in the developing forebrain, diencephalon, midbrain/hindbrain boundary and spinal cord. Both genes are expressed from E7 in opposite gradients in primitive streak mesoderm. Subsequently their transcripts are localized to regions of mesenchyme/epithelial interaction in the palate, heart, limbs, anal and urogenital region.

Primary structure, chromosomal mapping, expression and transcriptional activity of murine hepatocyte nuclear factor 4gamma.

We demonstrate the presence of a new member of the orphan nuclear receptor hepatocyte nuclear factor 4 (HNF4) subfamily in mouse which is genetically distinct from the previously characterized mouse HNF4alpha gene. The new member of the HNF4 subfamily shows highest amino acid identity, similar tissue distribution and syntenous chromosomal localization to the recently described human HNF4gamma (NR2A2), we therefore classify it as mouse HNF4gamma (mHNF4gamma). A combination of RT-PCR and immunohistochemical analysis showed expression of mHNF4gamma mRNA and protein in the endocrine pancreas, testes, kidney and gut. By co-transfection experiments, we show that mHNF4gamma is able to activate transcription, acting through binding sites that have been previously characterized as HNF4alpha binding sites. The presence of HNFgamma in human and mouse implies that a complex transcriptional network exists in higher vertebrates involving a number of HNF4 members with overlapping yet distinct function and tissue distribution.

A novel murine PKA-related protein kinase involved in neuronal differentiation.

Members of the cAMP-dependent second-messenger pathway have been described as regulators of cellular growth and differentiation and were consequently implicated in a variety of embryogenic processes including brain development. Moreover, recent data suggest an indispensable role for cAMP-dependent protein kinases (PKAs) in neuronal differentiation and synaptic plasticity. Using a degenerate primer-based approach, we have identified a novel murine gene closely related to the human cAMP-dependent protein kinase PRKX on Xp22.3. This gene (Pkare) was mapped to the region near the centromere of the murine X chromosome and is expressed in a variety of adult organs including kidney, liver, spleen, testis, ovary, lung, heart, and brain. Antisense in situ hybridization on staged mouse embryos revealed a highly distinctive expression pattern during neuronal development, with elevated Pkare expression observed only in differentiating neurons within the first ganglion, the dorsal root ganglia, and the mantle layer of the telencephalon. Based on the close relationship with the catalytic PKA subunits and its distinct expression in differentiating neuronal cells, Pkare might represent a novel component of the cAMP-regulated pathways involved in brain development and function.

Dickkopf genes are co-ordinately expressed in mesodermal lineages.

Dickkopf-1 (dkk-1) is member of a novel family of secreted proteins and functions in head induction during Xenopus embryogenesis, acting as a potent inhibitor of Wnt signalling. Here we report: (1) the isolation of two additional murine members of the dkk family, dkk-2 and dkk-3; and (2) analysis of adult and embryonic gene expression of mouse dkk-1,-2, and -3, Xenopus dkk-1 as well as chicken dkk-3. Comparative developmental analyses of the dkk-1, dkk-2 and dkk-3 in mice indicate that these genes are both temporally and spatially regulated. They define overlapping deep domains in mesenchymal lineages suggesting a co-ordinated mode of action. All dkks show distinct and elevated expression patterns in tissues that mediate epithelial- mesenchyme transformations suggesting that they may participate in heart, tooth, hair and whisker follicle, limb and bone induction. In the limb buds expression of these genes are found in regions of programmed cell death. In a given organ, dkk-1 tends to be the earliest member expressed. Comparison with Xenopus dkk-1 and chicken dkk-3 shows evolutionarily conserved expression patterns. Our observations indicate that dkk genes constitute a new family of secreted proteins that may mediate inductive interactions between epithelial and mesenchymal cells.

Expression of the mouse Fkh1/Mf1 and Mfh1 genes in late gestation embryos is restricted to mesoderm derivatives.

We have compared the expression patterns of the mouse Forkhead homologue 1/ mesoderm/mesenchyme forkhead 1 (Fkh1/Mf1) gene with that of the highly related winged helix gene Mfh1 in late gestation mouse embryos. Transcripts for both genes are restricted to derivatives of the mesoderm. Co-expression was found in cartilage primordia of the head, ribs, vertebra and bones. However, in several structures analyzed, Fkh1/Mf1 signals are lower in the inner layers of the developing cartilage than those of Mfh1.

SHOT, a SHOX-related homeobox gene, is implicated in craniofacial, brain, heart, and limb development.

Deletion of the SHOX region on the human sex chromosomes has been shown to result in idiopathic short stature and proposed to play a role in the short stature associated with Turner syndrome. We have identified a human paired-related homeobox gene, SHOT, by virtue of its homology to the human SHOX and mouse OG-12 genes. Two different isoforms were isolated, SHOTa and SHOTb, which have identical homeodomains and share a C-terminal 14-amino acid residue motif characteristic for craniofacially expressed homeodomain proteins. Differences between SHOTa and b reside within the N termini and an alternatively spliced exon in the C termini. In situ hybridization of the mouse equivalent, OG-12, on sections from staged mouse embryos detected highly restricted transcripts in the developing sinus venosus (aorta), female genitalia, diencephalon, mes- and myelencephalon, nasal capsula, palate, eyelid, and in the limbs. SHOT was mapped to human chromosome 3q25-q26 and OG-12 within a syntenic region on chromosome 3. Based on the localization and expression pattern of its mouse homologue during embryonic development, SHOT represents a candidate for the Cornelia de Lange syndrome.

Dickkopf-1 is a member of a new family of secreted proteins and functions in head induction.

The Spemann organizer in amphibian embryos is a tissue with potent head-inducing activity, the molecular nature of which is unresolved. Here we describe dickkopf-1 (dkk-1), which encodes Dkk-1, a secreted inducer of Spemann's organizer in Xenopus and a member of a new protein family. Injections of mRNA and antibody indicate that dkk-1 is sufficient and necessary to cause head induction. dkk-1 s a potent antagonist of Wnt signalling, suggesting that dkk genes encode a family of secreted Wnt inhibitors.

Defective limbic system in mice lacking the tailless gene.

The gene tailless is a member of the superfamily of genes that encode transcription factors of the ligand-activated nuclear receptor type, and is expressed in the invertebrate and vertebrate brain. In mice, its transcripts are restricted to the periventricular zone of the forebrain, the site of origin of neurons and glia. Here we use homologous recombination to generate mice that lack a functional tailless protein. Homozygous mutant mice are viable at birth, indicating that tailless is not required for prenatal survival; however, adult mutant mice show a reduction in the size of rhinencephalic and limbic structures, including the olfactory, infrarhinal and entorhinal cortex, amygdala and dentate gyrus. Both male and female mice are more aggressive than usual and females lack normal maternal instincts. These animals therefore enable a molecular approach to be taken towards understanding the genetic architecture and morphogenesis of the forebrain.

Clustered arrangement of winged helix genes fkh-6 and MFH-1: possible implications for mesoderm development.

The 'winged helix' or 'forkhead' transcription factor gene family is defined by a common 100 amino acid DNA binding domain which is a variant of the helix-turn-helix motif. Here we describe the structure and expression of the mouse fkh-6 and MFH-1 genes. Both genes are expressed in embryonic mesoderm from the headfold stage onward. Transcripts for both genes are localised mainly to mesenchymal tissues, fkh-6 mRNA is enriched in the mesenchyme of the gut, lung, tongue and head, whereas MFH-1 is expressed in somitic mesoderm, in the endocardium and blood vessels as well as the condensing mesenchyme of the bones and kidney and in head mesenchyme. Both genes are located within a 10 kb region (in mouse chromosome 8 at 5.26 +/- 2.56 cM telomeric to Actsk1. The close physical linkage of these two winged helix genes is conserved in man, where the two genes map to chromosome 16q22-24. This tandem arrangement suggests the common use of regulatory mechanisms. The fkh-6/MFH-1 locus maps close to the mouse mutation amputated, which is characterised by abnormal development of somitic and facial mesoderm. Based on the expression patterns we suggest that a mutation in MFH-1, not fkh-6 is the possible cause for the amputated phenotype.

The mouse homolog of the region specific homeotic gene spalt of Drosophila is expressed in the developing nervous system and in mesoderm-derived structures.

The region specific homeotic gene spalt (sal) of Drosophila determines the specification of terminal segments. Its mutation leads to an incomplete transformation of terminal segments into trunk-like segments. The gene product is a zinc finger protein with a novel structure. We have isolated the mouse homolog of the Drosophila spalt gene (msal). The msal cDNA sequence is similar to its Drosophila counterpart in that it contains seven C2H2-type zinc finger motifs grouped into three pairs plus a single zinc finger closely linked to the middle pair. The two genes exhibit high sequence similarity in the zinc finger regions and to a lower extent in the putative transactivation domains. We have analysed the expression pattern of msal and show that it is expressed in the developing neuroectoderm of the brain, the inner ear and the spinal cord and in urogenital ridge-derived structures such as testis, ovaries and kidneys. A weaker and transient expression is seen in early embryos in the branchial arches and in tissues like the notochord, the limb buds and the heart. Given its role in Drosophila melanogaster and its strong sequence conservation, this expression pattern suggests an important role for msal in the development of the nervous system.

Regulation of Cre recombinase activity by the synthetic steroid RU 486.

To create a strategy for inducible gene targeting we developed a Cre-lox recombination system which responds to the synthetic steroid RU 486. Several fusions between Cre recombinase and the hormone binding domain (HBD) of a mutated human progesterone receptor, which binds RU 486 but not progesterone, were constructed. When tested in transient expression assays recombination activities of all fusion proteins were responsive to RU 486, but not to the endogenous steroid progesterone. However, the observed induction of recombination activity by the synthetic steroid varied between the different fusion proteins. The fusion with the highest activity in the presence of RU 486 combined with low background activity in the absence of the steroid was tested after stable expression in fibroblast and embryonal stem (ES) cells. We could demonstrate that its recombination activity was highly dependent on RU 486. Since the RU 486 doses required to activate recombination were considerably lower than doses displaying anti-progesterone effects in mice, this system could be used as a valuable tool for inducible gene targeting.

Expression of the winged helix genes fkh-4 and fkh-5 defines domains in the central nervous system.

The 'winged helix' or 'forkhead' transcription factor gene family is defined by a common 100 amino acid DNA binding domain which is a variant of the helix-turn-helix motif. Here we describe the structure and expression of the mouse fkh-4 and fkh-5 genes. The two genes encode proteins of 427 and 324 amino acids, respectively, with highly similar winged helix domains. Both genes are expressed in adjacent domains in the developing diencephalon from the headfold stage onward. Linkage analysis localised fkh-5 to chromosome 9 at 34.5 centiMorgans (cM) and fkh-4 to chromosome 19 at 10.5 cM. The potential relationship of the two genes to the mouse mutations staggerer and small thymus (for fkh-5) and muscle deficient (for fkh-4) is discussed.

The mouse fkh-2 gene. Implications for notochord, foregut, and midbrain regionalization.

The "winged helix" or "forkhead" transcription factors comprise a large gene family whose members are defined by a common 100-amino acid DNA binding domain. Here we describe the structure and expression of the mouse fkh-2 gene, which encodes a protein of 48 kDa with high similarity to other winged helix transcription factors within the DNA binding region, but unique potential transactivation domains. The gene is encoded by a single exon and is expressed in headfold stage embryos in the notochord, the anterior neuroectoderm, and a few cells of the definite endoderm. This expression becomes restricted to the anteriormost portions of the invaginating foregut and the developing midbrain. From day 11.5 of gestation onward, fkh-2 transcripts are restricted to the midbrain and become progressively localized to the red nuclei as the sole site of expression. The fkh-2 gene maps to chromosome 19B and is a candidate gene for the mouse mutation mdf (muscle-deficient) which is characterized by nervous tremors and degeneration of the hindlimb muscles. Although the expression patterns of the fkh-2 gene and another winged helix protein, HNF-3 beta, are overlapping in early stages of gestation and although the promoter of the fkh-2 gene contains a HNF-3 binding site, we demonstrate that the activation of the fkh-2 gene is independent of HNF-3 beta.

Targeted disruption of the glucocorticoid receptor gene blocks adrenergic chromaffin cell development and severely retards lung maturation.

The role of the glucocorticoid receptor (GR) in glucocorticoid physiology and during development was investigated by generation of GR-deficient mice by gene targeting. GR -/- mice die within a few hours after birth because of respiratory failure. The lungs at birth are severely atelectatic, and development is impaired from day 15.5 p.c. Newborn livers have a reduced capacity to activate genes for key gluconeogenic enzymes. Feedback regulation via the hypothalamic-pituitary-adrenal axis is severely impaired resulting in elevated levels of plasma adrenocorticotrophic hormone (15-fold) and plasma corticosterone (2.5-fold). Accordingly, adrenal glands are enlarged because of hypertrophy of the cortex, resulting in increased expression of key cortical steroid biosynthetic enzymes, such as side-chain cleavage enzyme, steroid 11 beta-hydroxylase, and aldosterone synthase. Adrenal glands lack a central medulla and synthesize no adrenaline. They contain no adrenergic chromaffin cells and only scattered noradrenergic chromaffin cells even when analyzed from the earliest stages of medulla development. These results suggest that the adrenal medulla may be formed from two different cell populations: adrenergic-specific cells that require glucocorticoids for proliferation and/or survival, and a smaller noradrenergic population that differentiates normally in the absence of glucocorticoid signaling.

The mouse homolog of the orphan nuclear receptor tailless is expressed in the developing forebrain.

The Drosophila tailless gene is a member of the orphan nuclear receptor subfamily. In Drosophila, the tailless gene is required for pattern formation in embryonic poles. During development, tailless is activated in the termini of the embryo in response to the torso receptor tyrosine kinase signal transduction cascade. Recessive mutations of tailless result in abnormalities in anterior portions of the head and in all structures posterior to the eighth abdominal segment. Localised expression of tailless is required in combination with a second terminal gene, huckebein, to control the expression of downstream genes. We have isolated a mouse homolog of the Drosophila tailless gene, which shows considerable homology in the DNA-binding domain suggesting that the respective proteins bind similar recognition sequences. Although the ligand-binding domain shows features in common with the tailless ligand domain, it also shares conserved amino acid stretches with other orphan nuclear receptors, the human ovalbumin upstream binding protein transcription factors (hCOUP-TF I and II). We have analysed the expression of taillees in mice, and show that it is specifically localised to the developing forebrain from day 8 p.c. and in dorsal midbrain from day 8.75 p.c. To define the anterior and posterior boundaries of expression, we compared the expression pattern of tailless to those of other forebrain markers, including distal-less (Dlx1), brain factor 1 (BF1), and the orthodenticle genes (Otx1 and Otx2). In addition to the developing forebrain, these genes show dynamic patterns of expression in two structures whose development requires inductive signals from the forebrain: the eye and the nose. These results suggest that the mouse taillees gene may be required to pattern anterior brain differentiation.

Molecular genetic analysis of glucocorticoid signaling during mouse development.

Glucocorticoids are important in a number of developmental processes in mammals around birth. The pathway of gluconeogenesis is activated in liver shortly after birth due to the combined effects of glucocorticoids and glucagon. We have defined the essential cis-regulatory elements directing hormone-dependent liver-specific expression of the gene for tyrosine aminotransferase, a key gluconeogenic enzyme. The hormone response elements synergize with cell-type specific elements. In the case of glucocorticoids, the glucocorticoid-dependent enhancer is composed of the glucocorticoid response element and binding sites for liver cell-enriched transcription factors, in particular hepatocyte nuclear factor-3. The dependence of the respective enhancer motifs on each other restricts the hormonal activation of the tyrosine aminotransferase gene in liver in response to a hormonal signal. To further understand the role of glucocorticoid signaling via the type II glucocorticoid receptor (GR) in the perinatal period and earlier during development, we have studied the expression of the mouse GR gene. Expression of the gene is controlled by at least three promoters, one of which is only active in T-lymphocytes. Expression of GR mRNA has been detected as early as day 9.5 of mouse development. To specifically address the role of glucocorticoid signaling via the GR during development, we have disrupted the GR gene by homologous recombination in mouse embryonic stem cells. The majority of GR mutants die shortly after birth and analysis so far has revealed defects in lung, liver, and adrenal function.

Characterization of the mouse HNF-4 gene and its expression during mouse embryogenesis.

Hepatocyte nuclear factor 4 (HNF-4) is a member of the nuclear receptor gene superfamily with unknown ligand. It has been assumed to play an important role in the regulation of gene expression in the liver. Here, we report the cloning and characterization of the mouse HNF-4 gene, as well as its expression during embryogenesis. The HNF-4 protein is encoded by ten exons. The gene structure is unique in the steroid receptor superfamily in that the second zinc finger is encoded by two exons. HNF-4 mRNA is expressed in a limited number of mouse adult tissues: liver, kidney, intestine, stomach and skin. HNF-4 could play an important role in the formation and function of visceral yolk sac and in the development of the liver and kidney since its mRNA, as determined by in situ hybridization, appears upon primary differentiation of these organs. As a first step in the study of the regulatory elements of the HNF-4 gene, we mapped the transcription start site and carried out DNase I hypersensitive site (HS) analysis over a region of approximately 22kb upstream of the gene. The complexity of the HSs suggests that multiple elements might contribute to the transcriptional regulation of the HNF-4 gene.