SummaryHox transcription factors (TFs) are essential for vertebrate development, but how these evolutionary conserved proteins function in vivo remains unclear. Because Hox proteins have notoriously low binding specificity, they are believed to bind with cofactors, mainly homeodomain TFs Pbx and Meis, to select their specific targets. We mapped binding of Meis, Pbx, and Hoxa2 in the branchial arches, a series of segments in the developing vertebrate head. Meis occupancy is largely similar in Hox-positive and -negative arches. Hoxa2, which specifies second arch (IIBA) identity, recognizes a subset of Meis prebound sites that contain Hox motifs. Importantly, at these sites Meis binding is strongly increased. This enhanced Meis binding coincides with active enhancers, which are linked to genes highly expressed in the IIBA and regulated by Hoxa2. These findings show that Hoxa2 operates as a tissue-specific cofactor, enhancing Meis binding to specific sites that provide the IIBA with its anatomical identity.

During development, a specific subset of ventral spinal cord precursors called pMN cells produces first motor neurons and then oligodendrocyte progenitor cells (OPCs), which migrate, divide and differentiate as myelinating oligodendrocytes. pMN cells express the Olig2 transcription factor and Olig2 function is necessary for formation of spinal motor neurons and OPCs. In the hindbrain and midbrain, distinct classes of visceral, branchiomotor and somatic motor neurons are organized as discrete nuclei, and OPCs are broadly distributed. Mouse embryos deficient for Olig2 function lack somatic motor neurons and OPCs, but it is not clear whether this reflects a common origin for these cells, similar to spinal cord, or independent requirements for Olig2 function in somatic motor neuron and OPC development. We investigated cranial motor neuron and OPC development in zebrafish and found, using a combination of transgenic reporters and cell type specific antibodies, that somatic abducens motor neurons and a small subset of OPCs arise from common olig2 ϩ neuroepithelial precursors in rhombomeres r5 and r6, but that all other motor neurons and OPCs do not similarly develop from shared pools of olig2 ϩ precursors. In the absence of olig2 function, r5 and r6 precursors remain in the cell cycle and fail to produce abducens motor neurons, and OPCs are entirely lacking in the hindbrain. These studies, therefore, reveal both common and independent roles for olig2 in development of somatic motor neurons and oligodendrocytes of the hindbrain.

Proper functioning of the vertebrate central nervous system requires the precise positioning of many neuronal cell types. This positioning is established during early embryogenesis when gene regulatory networks pattern the neural tube along its anteroposterior and dorsoventral axes. Dorsoventral patterning of the embryonic neural tube gives rise to multiple progenitor cell domains that go on to differentiate unique classes of neurons and glia. While the genetic program is reasonably well understood for some lineages, such as ventrally derived motor neurons and glia, other lineages are much less characterized. Here we show that prdm12b, a member of the PR domain containing-family of transcriptional regulators, is expressed in the p1 progenitor domain of the zebrafish neural tube in response to Sonic Hedgehog signaling. We find that disruption of prdm12b function leads to dorsal expansion of nkx6.1 expression and loss of p1-derived eng1b-expressing V1 interneurons, while the adjacent p0 and p2 domains are unaffected. We also demonstrate that prdm12b-deficient fish exhibit an abnormal touch-evoked escape response with excessive body contractions and a prolonged response time, as well as an inability to coordinate swimming movements, thereby revealing a functional role for V1 interneurons in locomotor circuits. We conclude that prdm12b is required for V1 interneuron specification and that these neurons control swimming movements in zebrafish.

BackgroundThe developing vertebrate hindbrain is transiently segmented into rhombomeres by a process requiring Hox activity. Hox genes control specification of rhombomere fates, as well as the stereotypic differentiation of rhombomere-specific neuronal populations. Accordingly, germ line disruption of the paralog group 1 (PG1) Hox genes Hoxa1 and Hoxb1 causes defects in hindbrain segmentation and neuron formation in mice. However, antisense-mediated interference with zebrafish hoxb1a and hoxb1b (analogous to murine Hoxb1 and Hoxa1, respectively) produces phenotypes that are qualitatively and quantitatively distinct from those observed in the mouse. This suggests that PG1 Hox genes may have species-specific functions, or that anti-sense mediated interference may not completely inactivate Hox function in zebrafish.ResultsUsing zinc finger and TALEN technologies, we disrupted hoxb1a and hoxb1b in the zebrafish germ line to establish mutant lines for each gene. We find that zebrafish hoxb1a germ line mutants have a more severe phenotype than reported for Hoxb1a antisense treatment. This phenotype is similar to that observed in Hoxb1 knock out mice, suggesting that Hoxb1/hoxb1a have the same function in both species. Zebrafish hoxb1b germ line mutants also have a more severe phenotype than reported for hoxb1b antisense treatment (e.g. in the effect on Mauthner neuron differentiation), but this phenotype differs from that observed in Hoxa1 knock out mice (e.g. in the specification of rhombomere 5 (r5) and r6), suggesting that Hoxa1/hoxb1b have species-specific activities. We also demonstrate that Hoxb1b regulates nucleosome organization at the hoxb1a promoter and that retinoic acid acts independently of hoxb1b to activate hoxb1a expression.ConclusionsWe generated several novel germ line mutants for zebrafish hoxb1a and hoxb1b. Our analyses indicate that Hoxb1 and hoxb1a have comparable functions in zebrafish and mouse, suggesting a conserved function for these genes. In contrast, while Hoxa1 and hoxb1b share functions in the formation of r3 and r4, they differ with regards to r5 and r6, where Hoxa1 appears to control formation of r5, but not r6, in the mouse, whereas hoxb1b regulates formation of r6, but not r5, in zebrafish. Lastly, our data reveal independent regulation of hoxb1a expression by retinoic acid and Hoxb1b in zebrafish.

The embryonic vertebrate neural tube is divided along its dorsoventral (DV) axis into eleven molecularly discrete progenitor domains. Each of these domains gives rise to distinct neuronal cell types; the ventral-most six domains contribute to motor circuits, while the five dorsal domains contribute to sensory circuits. Following the initial neurogenesis step, these domains also generate glial cell types—either astrocytes or oligodendrocytes. This DV pattern is initiated by two morphogens—Sonic Hedgehog released from notochord and floor plate and Bone Morphogenetic Protein produced in the roof plate—that act in concentration gradients to induce expression of genes along the DV axis. Subsequently, these DV-restricted genes cooperate to define progenitor domains and to control neuronal cell fate specification and differentiation in each domain. Many genes involved in this process have been identified, but significant gaps remain in our understanding of the underlying genetic program. Here we review recent work identifying members of the Prdm gene family as novel regulators of DV patterning in the neural tube. Many Prdm proteins regulate transcription by controlling histone modifications (either via intrinsic histone methyltransferase activity, or by recruiting histone modifying enzymes). Prdm genes are expressed in spatially restricted domains along the DV axis of the neural tube and play important roles in the specification of progenitor domains, as well as in the subsequent differentiation of motor neurons and various types of interneurons. Strikingly, Prdm proteins appear to function by binding to, and modulating the activity of, other transcription factors (particularly bHLH proteins). The identity of key transcription factors in DV patterning of the neural tube has been elucidated previously (e.g. the nkx, bHLH and pax families), but it now appears that an additional family is also required and that it acts in a potentially novel manner.