Mice with either diminished or elevated levels of anti-Müllerian hormone have decreased litter sizes.

Anti-Müllerian hormone (AMH) is both a gonadal hormone and a putative paracrine regulator of neurons, the uterus, and the placenta. A mouse line with neuronal expression of AMH (Thy1.2-AMH) was generated to examine the role of paracrine AMH in the brain. The mice had normal behavior, but unexpectantly AMH was present in the circulation of the transgenic mice. Thy1.2-AMHTg/0 studs sired pups with a normal frequency, when mated with wild-type dams. In stark contrast, Thy1.2-AMHTg/0 dams rarely gave birth, with evidence of spontaneous midgestational abortion. This leads to the hypothesis that AMH influences the capacity of dams to carry concepti to term. This hypothesis was tested by mating AMH-deficient (Amh-/-), Thy1.2-AMHTg/0, and wild-type dams when 49-, 80-, and 111 days old, using proven wild-type studs. The litter sizes from the first two matings and the number of fetuses present on the 10th day of gestation of the third mating were recorded. Thy1.2-AMHTg/0 dams carried near normal numbers of midterm fetuses, but typically produced no pups, indicating that extensive late resorption of fetuses was occurring. Amh-/- dams exhibited a lesser reduction in litter size than the Thy1.2-AMHTg/0 dams, with no evidence of enhanced loss of fetuses. In conclusion, this study provides the first evidence that high AMH levels can cause a miscarriage phenotype and that the absence of AMH affects reproductive output.

The type 2 anti-Müllerian hormone receptor has splice variants that are dominant-negative inhibitors.

Anti-Müllerian hormone (AMH) has both paracrine and hormonal actions that occur at different AMH concentrations, and in cells with different densities of its specific receptor (Amhr2). This diversity is not explained by canonical AMH signaling. We report that Amhr2 has two splice variants: Amhr2Δ2 (AMH binding site) and Amhr2Δ9/10 (kinase domain). Both spliced variants inhibit AMH signaling in a reporter assay. The mRNA for the spliced variants was relatively less abundant than Amhr2 mRNA in all tissues. This suggests that the physiological function(s) of the receptor variants may be restricted to specific cellular/subcellular sites and/or to the transport of AMH.

Myocardial deletion of Smad4 using a novel α skeletal muscle actin Cre recombinase transgenic mouse causes misalignment of the cardiac outflow tract.

SMAD4 acts as the converging point for TGFß and BMP signaling in heart development. Here, we investigated the role of SMAD4 in heart development using a novel α skeletal muscle actin Cre recombinase (MuCre) transgenic mouse strain. Lineage tracing using MuCre/ROSA26(LacZ) reporter mice indicated strong Cre-recombinase expression in developing and adult heart and skeletal muscles. In heart development, significant MuCre expression was noted at E11.5 in the atrial, ventricular, outflow tract and atrioventricular canal myocardium, but not in the endocardial cushions. MuCre-driven conditional deletion of Smad4 in mice caused double outlet right ventricle (DORV), ventricular septal defect (VSD), impaired trabeculation and thinning of ventricular myocardium, and mid-gestational embryonic lethality. In conclusion, MuCre mice effectively delete genes in both heart and skeletal muscles, thus enabling the discovery that myocardial Smad4 deletion causes misalignment of the outflow tract and DORV.

Müllerian inhibiting substance contributes to sex-linked biases in the brain and behavior.

Many behavioral traits and most brain disorders are common to males and females but are more evident in one sex than the other. The control of these subtle sex-linked biases is largely unstudied and has been presumed to mirror that of the highly dimorphic reproductive nuclei. Sexual dimorphism in the reproductive tract is a product of Müllerian inhibiting substance (MIS), as well as the sex steroids. Males with a genetic deficiency in MIS signaling are sexually males, leading to the presumption that MIS is not a neural regulator. We challenge this presumption by reporting that most immature neurons in mice express the MIS-specific receptor (MISRII) and that male Mis(-/-) and Misrii(-/-) mice exhibit subtle feminization of their spinal motor neurons and of their exploratory behavior. Consequently, MIS may be a broad regulator of the subtle sex-linked biases in the nervous system.

Mice with disrupted TGFbeta signaling have normal cerebella development, but exhibit facial dysmorphogenesis and strain-dependent deficits in their body wall.

The transforming growth factor betas (TGFbetas) are context-dependent regulators of neurons in vitro, but their physiological functions in the brain are unclear. Haploinsufficiency of either Tgfbeta1 or Tgfbeta2 leads to age-related deterioration of neurons, but the development of the brain is normal in the full absence of either of these genes. However, some individuals with mis-sense mutations of TGFbeta receptors are mentally retarded, suggesting that the TGFbeta isoforms can compensate for each other during brain development. This possibility was tested by generating mice (NSE x PTR) with neuron-specific expression of a dominant-negative inhibitor of TGFbeta signaling. The NSE x PTR mice with a FVBxC57Bl/6 genetic background were viable and developed normally despite strong neuronal expression of the inhibitor of TGFbeta signaling. Their cerebella were of normal size and contained normal numbers of neurons. When the genetic background of the mice was changed to C57BL/6, the phenotype of the mice became neonatal lethal, with the neonates exhibiting various malformations. The malformations correlated with sites of non-neuronal expression of the transgenes and included facial dysmorphogenesis, incomplete closure of the ventral body wall and absence of intestinal motility. The C57BL/6 Tgfbm1-3 alleles, which modulate the phenotype of Tgfbeta1(-/-) mice, were not major determinants of the NSE x PTR phenotype. The data suggest that the development of the cerebellum is insensitive to the level of TGFbeta signaling, although this may be dependent on the genetic background.

The structure of the TGF-beta latency associated peptide region determines the ability of the proprotein convertase furin to cleave TGF-betas.

The TGF-beta family members are generated as latent pre-pro-polypeptides. The active mature peptides are cleaved from the latent forms by cellular proteases. TGF-beta 1, for instance, is predominantly processed by a substilisin-like proprotein convertase, furin. TGF-beta 2 has a consensus cleavage site for furin and therefore has been presumed to be cleaved by furin. However, TGF-beta 2 is often secreted as the latent form, which appears to be inconsistent with its postulated sensitivity to furin. We report here that both the regular (short) form of TGF-beta2 and its spliced variant with an additional exon (long form) are insensitive to furin. NIH 3T3 and CHO cells were transfected with expression vectors containing the short or long form of TGF-beta 2 or a chimeric TGF-beta consisting of the TGF-beta1 LAP region, the TGF-beta 2 cleavage site and the TGF-beta 2 mature peptide. The constructs included a c-myc epitope tag in the N-terminal region of the mature peptide. The TGF-betas produced by the transfected cells were analyzed with Western blots and immunocytochemistry. The intracellular proteins harvested from these cells were incubated with furin. Furin only inefficiently cleaved both the long and short forms of TGF-beta 2, but efficiently processed the chimeric TGF-beta. This indicates that the insensitivity of both forms of TGF-beta 2 to furin is a consequence of the tertiary structure of their LAP regions rather than their cleavage site. This differential processing of TGF-beta1 and -beta 2 may be part of the mechanism that generates isoform-specific functions of the TGF-betas.

BMP6 is axonally transported by motoneurons and supports their survival in vitro.

The regulation of motoneuron survival is only partially elucidated. We have sought new survival factors for motoneuron by analyzing which receptors they produce. We report here that the type II bone morphogenetic receptor (BMPRII) mRNA is one of the most abundant receptor mRNAs in laser microdissected motoneurons. Motoneurons were intensely stained by an anti-BMPRII antibody, indicating the presence of BMPRII protein. One of its ligands (BMP6) supported the survival of motoneurons in vitro. BMP6 was produced by myotubes and mature Schwann cells and was retrogradely transported in mature motor axons. BMP6 thus joins a list of known Schwann-cell-derived regulators of motoneurons, which includes GDNF, CNTF, LIF and TGF-beta2. The control of the production of these factors by Schwann cells and the direction of their movement in motor axons is diverse. This suggests that the multiplicity of motoneuron factors is because cells use different factors to regulate different aspects of motoneuron function.

Transforming growth factor beta2 haploinsufficient mice develop age-related nigrostriatal dopamine deficits.

The transforming growth factor-betas (TGF-betas) regulate the induction of dopaminergic neurons and are elevated in the CSF of Parkinson's patients. We report here that mice with TGF-beta2 haploinsufficiency (TGF-beta2+/-) have subclinical defects in the dopaminergic neurons of their substantia nigra pars compacta. At 6 weeks of age, the TGF-beta2+/- mice had 12% fewer dopaminergic neurons than wild-type littermates. No additional loss of neurons occurred during the next 5 months, although striatal dopamine declined to 70% of normal. The level of 3,4-dihydroxphenylacetic acid was normal in the TGF-beta2+/- mice, indicating that a compensatory mechanism maintains dopamine stimulation of their striatum. The TGF-beta2+/- mice had normal sensitivity to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, despite having reduced levels of monoamine oxidase-B. These results raise the possibility that people with naturally low levels of TGF-beta2 may have less functional reserve in their nigrostriatal pathway, causing them to be at increased risk of developing Parkinson disease.

Mullerian inhibiting substance acts as a motor neuron survival factor in vitro.

The survival of motor neurons is controlled by multiple factors that regulate different aspects of their physiology. The identification of these factors is important because of their relationship to motor neuron disease. We investigate here whether Mullerian Inhibiting Substance (MIS) is a motor neuron survival factor. We find that motor neurons from adult mice synthesize MIS and express its receptors, suggesting that mature motor neurons use MIS in an autocrine fashion or as a way to communicate with each other. MIS was observed to support the survival and differentiation of embryonic motor neurons in vitro. During development, male-specific MIS may have a hormone effect because the blood-brain barrier has yet to form, raising the possibility that MIS participates in generating sex-specific differences in motor neurons.

Transgenic mice carrying a tetracycline-inducible, truncated transforming growth factor beta receptor (TbetaRII).

The transforming growth factor-betas (TGFbetas) have multiple roles, making genetic analysis of their functions difficult. We therefore developed transgenic mouse lines to disrupt TGFbeta signaling using a mechanism that is inducible, reversible, and cell-type specific. The transgenic mouse lines carry an EGFP-pBi-DeltaTbetaRII construct (PTR). The DeltaTbetaRII element codes for a dominant-negative receptor that is known to disrupt TGFbeta signaling. The DeltaTbetaRII has a c-myc tag. The transgene was silent in the PTR mice, with expression of both EGFP and DeltaTbetaRII occurring when the PTR mice were crossed with mice that express the tetracycline transactivator (CMV-tTA). The expression of EGFP was repressed by the addition of doxycycline to the drinking water of the PTRxCMV-tTA mice. The PTR mice were then crossed with neuron-specific-tTA mice. Expression of the DeltaTbetaRII transgene in these mice led to an upregulation of native TGFbeta receptor expression, suggesting that neurons can modulate their responsiveness to TGFbetas.

Transforming growth factor-beta 2 causes an acute improvement in the motor performance of transgenic ALS mice.

Amyotrophic lateral sclerosis (ALS) is fatal disorder, characterized by the loss of motoneurons. The therapeutic potential of transforming growth factor-beta 2 (TGF-beta2) was examined using SOD1 mice. The SOD1 mice were treated with TGF-beta2 by repeated intraperitoneal injections. The highest dose of TGF-beta2 caused a rapid and marked improvement in the motor performance of the mice. This improvement lasted for between 2 and 3 weeks after which the TGF-beta2-treated mice rapidly deteriorated. At postmortem, the motoneurons in the TGF-beta2-treated SOD1 mice exhibited a large hypertrophy of their nucleoli, nuclei, and axons. In contrast, TGF-beta2 did not reverse the mitochondrial pathology. This may explain why the beneficial effects of TGF-beta2 and other growth factor on SOD1 mice are transient: TGF-beta2 is stimulating the motoneurons metabolic rate while one of their key metabolic organelles is damaged. Consequently, TGF-beta2 may be therapeutic for the forms ALS, with minimal mitochondrial involvement.

Transport of transforming growth factor-beta 2 across the blood-brain barrier.

The blood-brain barrier acts as an interface between the brain and body through a combination of restrictive mechanisms and transport processes. Substances essential for brain function pass through the barrier either by passive diffusion or by active transport. We report here that [125I]-transforming growth factor-beta2 (TGF-beta2) passes through the blood-brain barrier and blood-nerve barriers, after intravenous, intraperitoneal or intramuscular injections. The entry of the [125I]-TGF-beta2 to the brain was rapid, saturable and inhibited by co-injection of unlabelled TGF-beta2. In contrast, co-injection of unlabelled TGF-beta2 increased the retention of [125I]-TGF-beta2 in the blood. The [125I]-TGF-beta2 transported into the brain was localised by autoradiography to the extracellular space, and was intact as judged by SDS-PAGE. The [125I]-TGF-beta2 was widely distributed throughout the brain, with the highest concentrations in the hypothalamus and nerves and the lowest in the cerebral hemispheres. The [125I]-TGF-beta2 had a half-life of 4 h in the brain. These results indicate that therapeutically relevant levels of TGF-beta2 reach the brain after peripheral administration of TGF-beta2.

Fetal and maternal transforming growth factor-beta 1 may combine to maintain pregnancy in mice.

One of the mysteries of pregnancy is why a mother does not reject her fetuses. Cytokine-modulation of maternal-fetal interactions is likely to be important. However, mice deficient in transforming growth factor-beta1 (TGF beta 1) and other cytokines are able to breed, bringing this hypothesis into question. The phenotype of TGF beta 1 null-mutant mice varies with genetic background. We report here that, in outbred mice, the loss of TGF beta 1-deficient embryos is influenced by the parity of their mother. This is consistent with the loss of mutants being due to immune rejection. An inbred line of TGF beta 1(+/-) mice that supported TGF beta 1-deficient fetuses had high levels of TGF beta 1 in their plasma. Analysis of the amniotic fluids in this line indicated that biologically relevant levels of maternal TGF beta 1 were present in the TGF beta 1(-/-) fetuses. These data are consistent with maternal and fetal TGF beta 1 interacting to maintain pregnancy, within immune-competent mothers.

Transforming growth factor beta 1 may regulate the stability of mature myelin sheaths.

The molecular mechanisms underlying peripheral neuropathies have only been partially elucidated. In particular, the regulatory factors that control the stability and turnover of mature myelin are largely unknown. Transforming growth factor beta 1 (TGF-beta1), and its associated receptors, are expressed by mature Schwann cells. On this basis, we postulated that TGF-beta1 may be an autocrine regulator of mature myelin. This hypothesis was tested by examining the ultrastructure of myelin in adult mice that have a null mutation of their TGF-beta1 gene. We report here that the myelin of these mice is grossly abnormal. At the nodes of Ranvier, the cytoplasmic collars of the Schwann cells were expanded and the myelin had a honeycomb appearance. Focal (tomacula-like) hypermyelin structures were observed in the internodal regions of a significant number of axons in mutant nerve, and were not observed in littermate controls. Axon diameters were within the normal range and no axonal pathology was evident in mutant nerve and macrophages were absent. Results imply that lack of TGF-beta1 may have a direct effect on Schwann cells. We suggest that TGF-beta1 may stabilise compact myelin via an autocrine mechanism.

The transforming growth factor-betas: multifaceted regulators of the development and maintenance of skeletal muscles, motoneurons and Schwann cells.

This review discusses the roles of the transforming growth factor-betas (TGF-betas) as part of a complex network that regulates the development and maintenance of the neuromuscular system. The actions of the TGF-betas often vary depending on which other growth factors are present, making it difficult to extrapolate results from in vitro experiments to the in vivo situation. A new approach has therefore been needed to understand the physiological functions of the TGF-betas. The behaviours (proliferation, fusion, apoptosis) of many of the cells in the neuromuscular system have a complex pattern which varies in space and time. The actions of growth factors in this system can thus be deduced based on how well their pattern of expression correlates with known cellular behaviours. Hypotheses based on this molecular anatomical evidence can then be further tested with genetically modified mice. From this type of evidence, we suggest that: (1) TGF-beta1 is an autocrine regulator of Schwann cells; (2) maternally-derived TGF-beta1 helps to suppress self and maternal immune attack; (3) TGF-beta2 regulates when and where myoblasts fuse to myotubes; (4) motoneuron survival is regulated by multiple sources of TGF-betas, with TGF-beta2 being the more important isoform. The concept of TGF-beta1 as a regulator of secondary myotube formation is not supported by either the location of the TGF-beta1 in developing muscles or by the phenotype of TGF-beta1-/- mice. The review concludes with a discussion of whether all of these of postulated functions can occur independently of each other, within the confines of the neuromuscular system.