Cindy C. Lu

Nodal signalling in the epiblast patterns the early mouse embryo

Shortly after implantation the mouse embryo comprises three tissue layers. The founder tissue of the embryo proper, the epiblast, forms a radially symmetric cup of epithelial cells that grows in close apposition to the extra-embryonic ectoderm and the visceral endoderm. This simple cylindrical structure exhibits a distinct molecular pattern along its proximal–distal axis. The anterior–posterior axis of the embryo is positioned later by coordinated cell movements that rotate the pre-existing proximal–distal axis. The transforming growth factor-β family member Nodal is known to be required for formation of the anterior–posterior axis. Here we show that signals from the epiblast are responsible for the initiation of proximal–distal polarity. Nodal acts to promote posterior cell fates in the epiblast and to maintain molecular pattern in the adjacent extra-embryonic ectoderm. Both of these functions are independent of Smad2. Moreover, Nodal signals from the epiblast also pattern the visceral endoderm by activating the Smad2-dependent pathway required for specification of anterior identity in overlying epiblast cells. Our experiments show that proximal–distal and subsequent anterior–posterior polarity of the pregastrulation embryo result from reciprocal cell–cell interactions between the epiblast and the two extra-embryonic tissues.

From fertilization to gastrulation: axis formation in the mouse embryo

Although much remains unknown about how the embryonic axis is laid down in the mouse, it is now clear that reciprocal interactions between the extraembryonic and embryonic lineages establish and reinforce patterning of the embryo. At early post-implantation stages, the extraembryonic ectoderm appears to impart proximal-posterior identity to the adjacent proximal epiblast, whereas the distal visceral endoderm signals to the underlying epiblast to restrict posterior identity as it moves anteriorward. At gastrulation, the visceral endoderm is necessary for specifying anterior primitive streak derivatives, which, in turn, pattern the anterior epiblast. Polarity of these extraembryonic tissues can be traced back to the blastocyst stage, where asymmetry has been linked to the point of sperm entry at fertilization.

Developmental Profiling of Spiral Ganglion Neurons Reveals Insights into Auditory Circuit Assembly

The sense of hearing depends on the faithful transmission of sound information from the ear to the brain by spiral ganglion (SG) neurons. However, how SG neurons develop the connections and properties that underlie auditory processing is largely unknown. We catalogued gene expression in mouse SG neurons from embryonic day 12, when SG neurons first extend projections, up until postnatal day 15, after the onset of hearing. For comparison, we also analyzed the closely related vestibular ganglion (VG). Gene ontology analysis confirmed enriched expression of genes associated with gene regulation and neurite outgrowth at early stages, with the SG and VG often expressing different members of the same gene family. At later stages, the neurons transcribe more genes related to mature function, and exhibit a dramatic increase in immune gene expression. Comparisons of the two populations revealed enhanced expression of TGFβ pathway components in SG neurons and established new markers that consistently distinguish auditory and vestibular neurons. Unexpectedly, we found that Gata3, a transcription factor commonly associated with auditory development, is also expressed in VG neurons at early stages. We therefore defined new cohorts of transcription factors and axon guidance molecules that are uniquely expressed in SG neurons and may drive auditory-specific aspects of their differentiation and wiring. We show that one of these molecules, the receptor guanylyl cyclase Npr2, is required for bifurcation of the SG central axon. Hence, our dataset provides a useful resource for uncovering the molecular basis of specific auditory circuit assembly events.

Gata3 Is a Critical Regulator of Cochlear Wiring

Spiral ganglion neurons (SGNs) play a key role in hearing by rapidly and faithfully transmitting signals from the cochlea to the brain. Identification of the transcriptional networks that ensure the proper specification and wiring of SGNs during development will lay the foundation for efforts to rewire a damaged cochlea. Here, we show that the transcription factor Gata3, which is expressed in SGNs throughout their development, is essential for formation of the intricately patterned connections in the cochlea. We generated conditional knock-out mice in which Gata3 is deleted after SGNs are specified. Cochlear wiring is severely disrupted in these animals, with premature extension of neurites that follow highly abnormal trajectories toward their targets, as shown using in vitro neurite outgrowth assays together with time-lapse imaging of whole embryonic cochleae. Expression profiling of mutant neurons revealed a broad shift in gene expression toward a more differentiated state, concomitant with minor changes in SGN identity. Thus, Gata3 appears to serve as an “intermediate regulator” that guides SGNs through differentiation and preserves the auditory fate. As the first auditory-specific regulator of SGN development, Gata3 provides a useful molecular entry point for efforts to engineer SGNs for the restoration of hearing.

Changes in Sef Levels Influence Auditory Brainstem Development and Function

During development of the CNS, secreted morphogens of the fibroblast growth factor (FGF) family have multiple effects on cell division, migration, and survival depending on where, when, and how much FGF signal is received. The consequences of misregulating the FGF pathway were studied in a mouse with decreased levels of the FGF antagonist Sef. To uncover effects in the nervous system, we focused on the auditory system, which is accessible to physiological analysis. We found that the mitogen-activated protein kinase pathway is active in the rhombic lip, a germinal zone that generates diverse types of neurons, including the cochlear nucleus complex of the auditory system. Sef is expressed immediately adjacent to the rhombic lip, overlapping with FGF15 and FGFR1, which is also present in the lip itself. This pattern suggests that Sef may normally function in non-rhombic lip cells and prevent them from responding to FGF ligand in the vicinity. Consistent with this idea, overexpression of Sef in chicks decreased the size of the auditory nuclei. Cochlear nucleus defects were also apparent in mice with reduced levels of Sef, with 13% exhibiting grossly dysmorphic cochlear nuclei and 26% showing decreased amounts of GFAP in the cochlear nucleus. Additional evidence for cochlear nucleus defects was obtained by electrophysiological analysis of Sef mutant mice, which have normal auditory thresholds but abnormal auditory brainstem responses. These results show both increases and decreases in Sef levels affect the assembly and function of the auditory brainstem.

Mutation of Npr2 Leads to Blurred Tonotopic Organization of Central Auditory Circuits in Mice

Tonotopy is a fundamental organizational feature of the auditory system. Sounds are encoded by the spatial and temporal patterns of electrical activity in spiral ganglion neurons (SGNs) and are transmitted via tonotopically ordered processes from the cochlea through the eighth nerve to the cochlear nuclei. Upon reaching the brainstem, SGN axons bifurcate in a stereotyped pattern, innervating target neurons in the anteroventral cochlear nucleus (aVCN) with one branch and in the posteroventral and dorsal cochlear nuclei (pVCN and DCN) with the other. Each branch is tonotopically organized, thereby distributing acoustic information systematically along multiple parallel pathways for processing in the brainstem. In mice with a mutation in the receptor guanylyl cyclase Npr2, this spatial organization is disrupted. Peripheral SGN processes appear normal, but central SGN processes fail to bifurcate and are disorganized as they exit the auditory nerve. Within the cochlear nuclei, the tonotopic organization of the SGN terminal arbors is blurred and the aVCN is underinnervated with a reduced convergence of SGN inputs onto target neurons. The tonotopy of circuitry within the cochlear nuclei is also degraded, as revealed by changes in the topographic mapping of tuberculoventral cell projections from DCN to VCN. Nonetheless, Npr2 mutant SGN axons are able to transmit acoustic information with normal sensitivity and timing, as revealed by auditory brainstem responses and electrophysiological recordings from VCN neurons. Although most features of signal transmission are normal, intermittent failures were observed in responses to trains of shocks, likely due to a failure in action potential conduction at branch points in Npr2 mutant afferent fibers. Our results show that Npr2 is necessary for the precise spatial organization typical of central auditory circuits, but that signals are still transmitted with normal timing, and that mutant mice can hear even with these deficits.