Expression of the LIM-homeodomain gene Lmx1a (dreher) during development of the mouse nervous system

The expression pattern of Lmx1a, a LIM-homeodomain gene disrupted in the dreher mouse neurological mutant, is described during development. Lmx1a is predominantly expressed in the developing nervous system from embryonic day E8.5 to adulthood, in restricted areas. Major expression domains include the dorsal midline (roof plate) of the neural tube, the cortical hem, the otic vesicles, the developing cerebellum and the notochord. The Lmx1a expression pattern is therefore well correlated with the various aspects of the phenotype of the dreher mutant mice.     

Dorsal patterning defects in the hindbrain, roof plate and skeleton in the dreher (dr(J)) mouse mutant

dreher is a spontaneous mouse mutation in which adult animals display a complex phenotype associated with hearing loss, neurological, pigmentation and skeletal abnormalities. During early embryogenesis, the neural tube of dreher mutants is abnormally shaped in the region of the rhomboencephalon, due to problems in the formation of a proper roof plate over the otic hindbrain. We have studied the expression of Hox/lacZ transgenic mouse strains in the dreher background and shown that primary segmentation of the neural tube is not altered in these mutants, although correct morphogenesis is affected resulting in misshapen rhombomeres. Neural crest derivatives from rhombomere 6, such as the glossopharyngeal ganglion, are defective, and the dorsal neural tube marker Wnt1 is absent from this segment. Selected trunk neural crest populations are also altered, as there is a lack of pigmentation in the thoracic region of mutant mice. Skeletal defects include abnormal cranial bones of neural crest origin, and improper fusion of the dorsal aspects of cervical and thoracic vertebrae. Taken together, the gene affected in the dreher mutant is responsible for correct patterning of the dorsal-most cell types of the neural tube, that is, the neural crest and the roof plate, in the hindbrain region. Axial skeletal defects could reflect inductive influence of the dorsal neural tube on proper fusion of the neural arches. It is possible that a common precursor population for both neural crest and roof plate is the cellular target of the dreher mutation.     

Roof plate-dependent patterning of the vertebrate dorsal central nervous system

In the vertebrate central nervous system (CNS), diverse cellular types are generated in response to inductive signals provided by specialized cellular groups that act as organizing centers. The roof plate is a critical dorsal signaling center that occupies the dorsal midline of the developing CNS along its entire anterior-posterior axis. During caudal neural tube development, the roof plate produces proteins of the Bmp and Wnt families controlling proliferation, specification, migration, and axon guidance of adjacent dorsal interneurons. Although primarily investigated in the developing spinal cord, a growing number of studies indicate that roof plate-derived signals are also critical for the patterning of dorsal structures in more rostral regions of CNS including the hindbrain, diencephalon and telencephalon. In this review, we discuss recent progress towards understanding the molecular and cellular mechanisms of roof plate-dependent patterning of the dorsal CNS.     

Signaling through BMP type 1 receptors is required for development of interneuron cell types in the dorsal spinal cord

During spinal cord development, distinct classes of interneurons arise at stereotypical locations along the dorsoventral axis. In this paper, we demonstrate that signaling through bone morphogenetic protein (BMP) type 1 receptors is required for the formation of two populations of commissural neurons, DI1 and DI2, that arise within the dorsal neural tube. We have generated a double knockout of both BMP type 1 receptors, Bmpr1a and Bmpr1b, in the neural tube. These double knockout mice demonstrate a complete loss of D1 progenitor cells, as evidenced by loss of Math1 expression, and the subsequent failure to form differentiated DI1 interneurons. Furthermore, the DI2 interneuron population is profoundly reduced. The loss of these populations of cells results in a dorsal shift of the dorsal cell populations, DI3 and DI4. Other dorsal interneuron populations, DI5 and DI6, and ventral neurons appear unaffected by the loss of BMP signaling. The Bmpr double knockout animals demonstrate a reduction in the expression of Wnt and Id family members, suggesting that BMP signaling regulates expression of these factors in spinal cord development. These results provide genetic evidence that BMP signaling is crucial for the development of dorsal neuronal cell types.     

Dorsal and intermediate neuronal cell types of the spinal cord are established by a BMP signaling pathway

We have studied the role of Bmp signaling in patterning neural tissue through the use of mutants in the zebrafish that disrupt three different components of a Bmp signaling pathway: swirl/bmp2b, snailhouse/bmp7 and somitabun/smad5. We demonstrate that Bmp signaling is essential for the establishment of the prospective neural crest and dorsal sensory Rohon-Beard neurons of the spinal cord. Moreover, Bmp signaling is necessary to limit the number of intermediate-positioned lim1+ interneurons of the spinal cord, as observed by the dramatic expansion of these prospective interneurons in many mutant embryos. Our analysis also suggests a positive role for Bmp signaling in the specification of these interneurons, which is independent of Bmp2b/Swirl activity. We found that a presumptive ventral signal, Hh signaling, acts to restrict the amount of dorsal sensory neurons and trunk neural crest. This restriction appears to occur very early in neural tissue development, likely prior to notochord or floor plate formation. A similar early role for Bmp signaling is suggested in the specification of dorsal neural cell types, since the bmp2b/swirl and bmp7/snailhouse genes are only coexpressed during gastrulation and within the tail bud, and are not found in the dorsal neural tube or overlying epidermal ectoderm. Thus, a gastrula Bmp2b/Swirl and Bmp7/Snailhouse-dependent activity gradient may not only act in the specification of the embryonic dorsoventral axis, but may also function in establishing dorsal and intermediate neuronal cell types of the spinal cord.     

Molecular and morphological characterization of neural tube defects in embryos of diabetic Swiss Albino mice

BACKGROUND AND RESULTS: Embryos from diabetic mice exhibit several forms of neural tube defects, including non-closure of the neural tube. In the present study, embryos collected at embryonic day 11.5 from diabetic pregnancies displayed open neural tube with architectural disruption of the surrounding tissues. The percentage of proliferating cells was found to be increased in the dorsal and ventral domains of the spinal neural tube of embryos from diabetic mice, indicating a defect in the proliferation index. We have analyzed the development of various cell types, including motoneurons, interneurons, oligodendrocytes and migrating neurons, as well as radial glial cells in the open neural tube using specific molecular markers. Immunofluorescence results revealed a significantly reduced number of Pax2+ interneurons and increased number of Isl-1+ motoneurons, as well as Olig2+ oligodendrocytes in the neural tube of embryos from diabetic mice as compared to controls. In addition, these embryos exhibited a decreased number of doublecortin positive migrating neurons and Glast/Blbp positive radial glial cells with shortened processes in the neural tube. Expression levels of several developmental control genes involved in the generation of different neuronal cell types (such as Shh, Ngn, Ngn2, Ascl1) were also found to be altered in the neural tube of embryos from diabetic mice. CONCLUSIONS: Overall, the open neural tube in embryos of diabetic mice exhibits defects in the specification of different cell types, including motoneurons and interneurons, as well as glial cells along the dorsoventral axis of the developing spinal cord. Although these defects are associated with altered expression of several development control genes, the exact mechanisms by which maternal diabetes contributes to these changes remain to be investigated.     

The homeodomain factor lbx1 distinguishes two major programs of neuronal differentiation in the dorsal spinal cord

Dorsal horn neurons in the spinal cord integrate and relay sensory information. Here, we show that the expression of the homeobox gene Lbx1 distinguishes two major neuronal classes generated in the dorsal spinal cord. The Lbx1(-) (class A) and Lbx1(+) (class B) neurons differ in their dependence on roof plate BMP signals for specification and settle in the deep and superficial dorsal horn, respectively. Lbx1 misexpression blocks the differentiation of class A neurons. Conversely, in Lbx1 mutant mice, class B neurons assume the identity of class A neurons. As a consequence, the morphology and neuronal circuitry of the dorsal horn are aberrant. We conclude that Lbx1 distinguishes two major neuronal classes in the dorsal spinal cord and is an important determinant of their distinct differentiation programs.     

Ptf1a determines GABAergic over glutamatergic neuronal cell fate in the spinal cord dorsal horn

Mutations in the human and mouse PTF1A/Ptf1a genes result in permanent diabetes mellitus and cerebellar agenesis. We show that Ptf1a is present in precursors to GABAergic neurons in spinal cord dorsal horn as well as the cerebellum. A null mutation in Ptf1a reveals its requirement for the dorsal horn GABAergic neurons. Specifically, Ptf1a is required for the generation of early-born (dI4, E10.5) and late-born (dIL(A), E12.5) dorsal interneuron populations identified by homeodomain factors Lhx1/5 and Pax2. Furthermore, in the absence of Ptf1a, the dI4 dorsal interneurons trans-fate to dI5 (Lmx1b(+)), and the dIL(A) to dIL(B) (Lmx1b(+);Tlx3(+)). This mis-specification of neurons results in a complete loss of inhibitory GABAergic neurons and an increase in the excitatory glutamatergic neurons in the dorsal horn of the spinal cord by E16.5. Thus, Ptf1a function is essential for GABAergic over glutamatergic neuronal cell fates in the developing spinal cord, and provides an important genetic link between inhibitory and excitatory interneuron development.     

Zic1 and Zic4 regulate zebrafish roof plate specification and hindbrain ventricle morphogenesis

During development, the lumen of the neural tube develops into a system of brain cavities or ventricles, which play important roles in normal CNS function. We have established that the formation of the hindbrain (4th) ventricle in zebrafish is dependent upon the pleiotropic functions of the genes implicated in human Dandy Walker Malformation, Zic1 and Zic4. Using morpholino knockdown we show that zebrafish Zic1 and Zic4 are required for normal morphogenesis of the 4th ventricle. In Zic1 and/or Zic4 morphants the ventricle does not open properly, but remains completely or partially fused from the level of rhombomere (r) 2 towards the posterior. In the absence of Zic function early hindbrain regionalization and neural crest development remain unaffected, but dorsal hindbrain progenitor cell proliferation is significantly reduced. Importantly, we find that Zic1 and Zic4 are required for development of the dorsal roof plate. In Zic morphants expression of roof plate markers, including lmx1b.1 and lmx1b.2, is disrupted. We further demonstrate that zebrafish Lmx1b function is required for both hindbrain roof plate development and 4th ventricle morphogenesis, confirming that roof plate formation is a critical component of ventricle development. Finally, we show that dorsal rhombomere boundary signaling centers depend on Zic1 and Zic4 function and on roof plate signals, and provide evidence that these boundary signals are also required for ventricle morphogenesis. In summary, we conclude that Zic1 and Zic4 control zebrafish 4th ventricle morphogenesis by regulating multiple mechanisms including cell proliferation and fate specification in the dorsal hindbrain.     

Zic1 promotes the expansion of dorsal neural progenitors in spinal cord by inhibiting neuronal differentiation

The role of Zic1 was investigated by altering its expression status in developing spinal cords. Zic genes encode zinc finger proteins homologous to Drosophila Odd-paired. In vertebrate neural development, they are generally expressed in the dorsal neural tube. Chick Zic1 was initially expressed evenly along the dorsoventral axis and its expression became increasingly restricted dorsally during the course of neurulation. The dorsal expression of Zic1 was regulated by Sonic hedgehog, BMP4, and BMP7, as revealed by their overexpressions in the spinal cord. When Zic1 was misexpressed on the ventral side of the chick spinal cord, neuronal differentiation was inhibited irrespective of the dorsoventral position. In addition, dorsoventral properties were not grossly affected as revealed by molecular markers. Concordantly, when Zic1 was overexpressed in the dorsal spinal cord in transgenic mice, we observed hypercellularity in the dorsal spinal cord. The transgene-expressing cells were increased in comparison to those of truncated mutant Zic1-bearing mice. Conversely, we observed a significant cell number reduction without loss of dorsal properties in the dorsal spinal cords of Zic1-deficient mice. Taken together, these findings suggest that Zic1 controls the expansion of neuronal precursors by inhibiting the progression of neuronal differentiation. Notch-mediated inhibition of neuronal differentiation is likely to act downstream of Zic genes since Notch1 is upregulated in Zic1-overexpressing spinal cords in both the mouse and the chick.     

Canonical BMP7 activity is required for the generation of discrete neuronal populations in the dorsal spinal cord

BMP activity is essential for many steps of neural development, including the initial role in neural induction and the control of progenitor identities along the dorsal-ventral axis of the neural tube. Taking advantage of chick in ovo electroporation, we show a novel role for BMP7 at the time of neurogenesis initiation in the spinal cord. Using in vivo loss-of-function experiments, we show that BMP7 activity is required for the generation of three discrete subpopulations of dorsal interneurons: dI1-dI3-dI5. Analysis of the BMP7 mouse mutant shows the conservation of this activity in mammals. Furthermore, this BMP7 activity appears to be mediated by the canonical Smad pathway, as we demonstrate that Smad1 and Smad5 activities are similarly required for the generation of dI1-dI3-dI5. Moreover, we show that this role is independent of the patterned expression of progenitor proteins in the dorsal spinal cord, but depends on the BMP/Smad regulation of specific proneural proteins, thus narrowing this BMP7 activity to the time of neurogenesis. Together, these data establish a novel role for BMP7 in primary neurogenesis, the process by which a neural progenitor exits the cell cycle and enters the terminal differentiation pathway.