PMC

(A) Scanning electron microscopy (SEM) views of the cochlea at P8: an overview of the apical turns of WT, Hoxb1^null and Hoxb1^lateCKO cochleae showing three orderly arrayed rows of outer hair cells (OHCs) and one row of inner hair cells (IHCs). Representative high magnification images illustrate stereocilia of hair bundles of single OHCs arranged according to their different lengths. Shape and organization of OHCs in apical regions are normal at this stage in both mutants. (B) SEM views of 3-month-old WT, Hoxb1^null and Hoxb1^lateCKO cochleae and representative higher magnification images of OHCs. In Hoxb1^null and Hoxb1^lateCKO cochleae, OHCs have lost their regular organization and fail to develop in some areas (white arrowheads). Moreover, in Hoxb1^null cochleae most stereocilia have completely lost their typical V-shaped morphology and their characteristic differences in lengths (arrows). OHCs are less severely affected in Hoxb1^lateCKO cochleae. IHC cilia appeared weakly disarranged (red arrowheads). (C) SEM views of 3-months-old WT and Hoxb2^ΔKO mutant cochleae and higher magnifications of representative OHCs. Note that, similarly to Hoxb1^lateCKO cochleae, Hoxb2 mutants have occasional missing OHCs (white arrowheads), disarranged IHC cilia (red arrowheads) and disorganized OHC stereocilia (arrows). (D) Histogram quantifying the percentage of OHC loss in controls, Hoxb1^null, Hoxb1^lateCKO and Hoxb2^ΔKO cochleae. While controls (n = 8) showed no OHC loss, in Hoxb1^null mutants (n = 6) 7.2±0.8% of OHCs were absent, whereas 3.1±0.9% and 1.3±0.5% were lost in Hoxb1^lateCKO (n = 6) and Hoxb2^ΔKO (n = 3) cochleae, respectively. Inter-genotype ANOVA p<0.001; Hoxb1^null versus WT: p<0.001; Hoxb1^lateCKO versus WT: p<0.001; Hoxb2^ΔKO versus WT: p = 0.02. Scale bars, 10 µm (A, B, C, left panels), 1 µm (A, B, C, right panels). See also .

(A) Schematic view of a brain coronal section indicates the insertion of a DiI or dextran crystal into the cochlea to label controlateral MOC neurons. (B) Retrogradely-labeled MOC axons normally project across the midline as a compact bundle (arrow). No MOC axons crossing the midline are retrogradely labeled in Hoxb1^null and Hoxb1^lateCKO brains (asterisks). (C) Similarly, MOC fibers fail to cross the midline in Hoxb2^ΔKO mutant brains (asterisk), whereas no obvious defect is observed in Hoxa2^null mutants (arrow). (D) Schematic view of the organ of Corti, showing terminal innervation of the OHCs by the MOC efferent neurons (arrows). (E) Transmission electron microscopy of OHCs in adults WT, Hoxb1^null and Hoxb1^lateCKO cochleae. In high magnification views, MOC terminals synapse on OHCs in WT and Hoxb1^lateCKO cochleae, even if at much reduced ratio (F); a sub-synaptic cisterna is visualized inside the OHC (arrowheads). No MOC terminals are detected in Hoxb1^null cochleae. (F) Histogram showing the ratio of the number of MOC synaptic contacts on OHC visualized in TEM experiments in controls, Hoxb1^null and Hoxb1^lateCKO mutants. MOC/OHCs ratio: WT (n = 3; 32 OHCs): 1.9±0.2; Hoxb1^lateCKO (n = 6; 64 OHCs): 0.2±0.2; Hoxb1^null (n = 4; 40 OHCs): 0.1±0.1. Inter-genotype ANOVA p<0.001; Hoxb1^lateCKO versus WT: p<0.001; Hoxb1^null versus WT: p<0.001. Scale bars, 400 µm (B, C), 10 µm (E, left panel of WT), 50 µm (E, left panels of Hoxb1^null and Hoxb1^lateCKO), 1 µm (E, right panels). See also and .

(A) Schematic view of a sagittal brain section indicating the YFP⁺ r4-derived nuclei and projections. A strong reduction of the YFP⁺ VLL nucleus (arrowhead) and projections (arrow) in Hoxb1 mutants is observed. In constitutive mutants the reduction is much more severe than in conditional mutants, as quantified in (D). Adjacent sagittal sections show no Hoxb2 and Hoxa2-expressing cells in Hoxb1^null mutants, whereas cells in the reduced VLL of Hoxb1^lateCKO still express Hoxb2 and Hoxa2. Adjacent sections of another P8 pup confirm reduction of the VLL and indicate persistence of Gata3- and Gad67-expressing cells in both Hoxb1 mutants. No ectopic expression of VGlut2 is detected in the VLL region. (B) The VLL is reduced in Hoxb2^ΔKO mutant pups, similarly to Hoxb1^lateCKO mutants, as indicated by expression of Gata3, Gad67, Hoxa2 and quantification in (D). (C) In contrast, Hoxa2^null mutants show no significant changes in the VLL position and size quantified in (E). The apparently bigger shape is due to the slightly oblique sections in mutant compared to WT brains. (D) Histogram showing the percentage of the VLL area size in WT (set up to 100%) and in the different genotypes as indicated on the y-axis. Mutants show statistically significant differences when compared to WT, or when Hoxb1^lateCKO and Hoxb2^ΔKO are compared to Hoxb1^null (inter-genotype comparison, ANOVA p<0.001; Hoxb1^null versus WT: p = 0.001; Hoxb1^lateCKO versus WT: p = 0.01; Hoxb2^ΔKO versus WT: p = 0.04; Hoxb1^lateCKO versus Hoxb1^null: p<0.001; Hoxb2^ΔKO versus Hoxb1^null: p = 0.003). However, no statistically significant difference is found between Hoxb1^lateCKO versus Hoxb2^ΔKO (p = 0.39). (E) Histogram showing the percentage of the VLL area size in WT and Hoxa2^null. No statistically significant difference is found (WT versus Hoxa2^null: p = 0.56). FBM, facial branchiomotor nucleus; VLL, nucleus of lateral lemniscus; IC, inferior colliculus; MG, medial geniculate nucleus. Scale bars, 200 µm. See also .

(A) Oblique P8 coronal sections show abnormal presence of YFP⁺ fibers projecting to the medial nuclei of the trapezoid body (MNTB) in Hoxb1^null and Hoxb1^lateCKO mutants. Right panels: enlarged views of the boxed areas to the left depicting abnormal YFP⁺ terminals (arrowheads) of labeled crossed fibers (arrows) surrounding cells of the MNTB (limited by solid line). (B) Schematic representation showing the position of the injected dextran at the level of the PVCN (asterisk). Normally, PVCN interneurons innervate contralateral MOC neurons, as indicated in the control coronal section (arrow). In Hoxb2^ΔKO mutants, projections originating from the PVCN target now AVCN-specific targets, such as the contralateral MNTB (cMNTB) (arrowhead) and the lateral superior olivary (LSO) nuclei. (C) Schematics summarizing the normal connectivity pattern of cochlear AVCN neurons towards the nuclei of the superior olivary complex (SOC) complex, and the abnormal presence of YFP⁺ neurons behaving like AVCN neurons when Hoxb1 or Hoxb2 are inactivated. (D) A subpopulation of AVCN-labeled axons project abnormally to the ipsilateral MNTB (see arrowhead) when Hoxa2 is conditionally inactivated in the Wnt1⁺ (rhombic lip) domain. Expression of the Slit receptor Rig1 (or Robo3) is decreased in the VCN, indicating that absence of Rig1 affects midline crossing of AVCN projections. (E) Schematics summarizing the abnormal axonal projections of AVCN neurons in Wnt::Cre;Hoxa2lox/lox mice after dextran injection in the AVCN. AVCN, anterior ventral cochlear nucleus; PVCN, posterior ventral cochlear nucleus; PVCN IN, PVCN interneurons; MOC, medial olivocochlear neurons; aes, anterior extramural stream. Scale bars: 400 µm (A left panels, B), 50 µm (A right panels).

(A) Ectopic YFP⁺ r4-derived cells (arrows) are observed in the cochlear microneuronal shell (mnsh) (limited by solid line) of P8 Hoxb1^null and Hoxb1^lateCKO mutant sagittal sections. These cells now express Pax6 indicating that they are granule cells (white arrowheads). (B) Hoxb2 expression is decreased in the PVCN of Hoxb1^null and in Hoxb1^lateCKO mutants (asterisks). On the contrary, Hoxa2 expression is increased and Atoh7, normally expressed at high levels only in the AVCN, is dramatically up-regulated in the PVCN of P8 Hoxb1 (B) and Hoxb2^ΔKO mutant pups (C) (arrows). Arrowheads in WT indicate Hoxa2 and Atoh7 low-expressing regions. (D) Formation of the AVCN is strongly affected in E18.5 Hoxa2^null brains, as seen by decreased expression of Atoh7 and Hoxb2 (arrows). (E) Summary schematic indicating that in the absence of Hoxb1 and Hoxb2, the PVCN (r4-derived in brown) has acquired AVCN-like features (r2/3-derived in yellow) and YFP⁺ cell (brown) contribute to the shell. (F) The dorsal-most regions of WT and Hoxb1^null hindbrains at r3 and r4 levels on adjacent coronal sections hybridized with Atoh1 and revealed by YFP epifluorescence (indicates r4 levels). Atoh1 is expressed in progenitors and differentiating cells migrating along the lateral ridge. The Atoh1-expressing domain is reduced in r4 compared to r3 in WT, whereas an enlarged Atoh1-expressing domain (arrowhead) is identified in r4 of Hoxb1^null embryos. (G) On adjacent coronal sections at E14.5, YFP⁺ (r4-derived) cells located more laterally, do not overlap with Atoh1 ⁺ cells in the presumptive cochlear nucleus (CN), which originates from the r2–r5 auditory lip. In the absence of Hoxb1, YFP⁺ cells invade the Atoh1 ⁺ domain, thus acquiring the Atoh1 fate of adjacent rhombomeres. Cb, cerebellum; CES, caudal extramigratory stream. Scale bars, 200 µm (A up panels, B, C, D), 50 µm (A bottom panels), 100 µm (F, G). See also .

(A) Representative ABR measurements of 3 month-old WT, Hoxb1^null and Hoxb1^lateCKO mice. Five ABR peaks are the normal sequential responses evoked by an auditory stimulus at the level of the auditory nerve and along the series of auditory nuclei of the brainstem. The threshold, the lowest intensity of sound at which the response is present, is higher in Hoxb1 mutants than WT mice. (B) Latencies, the time intervals between the stimulus and the diverse response peaks, are normal at different intensities of sound. (C) Mean (± SE) thresholds measured in dB SPL (Sound Pressure Level) for WT, Hoxb1^lateCKO and Hoxb1^null mice at 1, 3, 6, 9 and 12 months of age. The differences between WT, Hoxb1^lateCKO and Hoxb1^null groups are statistically significant at all ages (1 month: WT, n = 24, 41.2±0.5; Hoxb1^null, n = 25, 79.0±2.1; Hoxb1^lateCKO, n = 23, 58.3±2.8; 3 months: WT, n = 28, 41.2±0.4; Hoxb1^null, n = 23, 84.3±1.7; Hoxb1^lateCKO, n = 33, 63.0±2.4; 6 months: WT, n = 22, 45.2±2.5; Hoxb1^null, n = 17, 96.5±2.6; Hoxb1^lateCKO, n = 25, 72.8±2.7; 9 months: WT, n = 20, 51.2±2.5, Hoxb1^null, n = 14, 102.1±3.1, Hoxb1^lateCKO, n = 17, 77.9±3.1; 12 months: WT, n = 10, 56±4, Hoxb1^null, n = 12, 108.9±3.0, Hoxb1^lateCKO, n = 14, 82.1±4.6; ANOVA p<0.001; post hoc t-test p<0.001; WT versus Hoxb1^null; WT versus Hoxb1^lateCKO: p<0.001 for all stages). A progressive increase of hearing thresholds in all groups with age is more prominent in Hoxb1^null and Hoxb1^lateCKO mutants than in the control mice. (D) Mean (± SE) thresholds of ABR for 3-month-old control and Hoxb2^ΔKO mice. The differences between the two groups are statistically significant (WT, n = 5, 52±1.3; Hoxb2^ΔKO, n = 5, 69±5.7; t-test p = 0.013); the threshold increase is similar to that of Hoxb1^lateCKO mice at the same age. (E) ABR of 1 month-old WT and Hoxb1^null mice put in an acoustically isolated environment from birth. Auditory thresholds are increased (WT: 48.9±0.2; Hoxb1^null: 84.2±0.4; p<0,01) similarly to those of non-acoustically isolated mutants (C). dB, decibel.

(A) The diagrams above the panels indicate the interactions between Hoxb1 and Hoxb2. While Hoxb1 auto-regulates its own expression in r4, it also binds to an Hoxb2 r4 enhancer to maintain Hoxb2 expression in r4. Hoxb2 maintains expression of Hoxb1 in r4. Crosses indicate loss of Hoxb1 protein in Hoxb1^null embryos and loss of the auto- and cross-regulatory loops in Hoxb1^lateCKO mutants. Lateral views of E8.5 to E9.25 embryos indicate that while Hoxb1 expression is still maintained in r4 (although at lower levels) of E8.75 Hoxb1^lateCKO mutants, r4 expression is completely abolished in E9.25 mutant embryos (arrowheads). Expression in the posterior region is still maintained at both ages (arrows). (B) Ventricular views of flat-mount preparations of E10.5 WT, Hoxb1^null and Hoxb1^lateCKO hindbrains hybridized with Hoxb2. Expression of Hoxb2 is strongly decreased (but not abolished) in r4 of Hoxb1^null and Hoxb1^lateCKO embryos, at similar levels to r3 (asterisks). R4 acquires an expression pattern of r3, as indicated by “r3”. Down-regulation of Hoxb2 in r4 can also be appreciated in mid-sagittal sections of mutant embryos. The line of cells expressing high levels of Hoxb2 denotes early post-mitotic cells (arrowhead). (C) Ventricular views of flat-mount preparations of E10.5 WT, Hoxb1^null and Hoxb1^lateCKO hindbrains hybridized with Hoxa2. Expression of Hoxa2 is increased in r4 and the characteristic Hoxa2 expression profile of r3 is now duplicated in r4 of Hoxb1^null and Hoxb1^lateCKO embryos supporting an r4 to r3 change of identity. The horizontal and vertical brackets indicate higher expression domains of Hoxa2, respectively in a large intermediate stripe and in a thinner lateral stripe of the sensory column, the presumptive auditory column. (D) On the left, expression of Hoxb1 indicates the position of r4 on sagittal sections. On the right, increased expression of Hoxa2 is detected in the ventricular and mantle layers of r4 at two different dorsal levels in Hoxb1^null embryos. In mutant embryos, the expression of Hoxa2 is maintained at levels comparable to r3 in the r4 mantle zone (mz) (i.e. post mitotic neurons) with respect to WT (arrows, see also insets). (E) In Hoxb2^ΔKO mutants, lack of Hoxb2 (indicated with a cross) results in failure to maintain Hoxb1 expression in r4. Sagittal and coronal views show that Hoxb1 protein is present in r4 of E8.75 embryos, but not maintained in E10.5 Hoxb2^ΔKO mutant embryos. (F) Flat-mounted hindbrain preparations hybridized with Hoxa2 show a duplication of r3 features in r4 in E10.5 Hoxb2^ΔKO mutant embryos. Hoxa2 expression levels are increased in r4 in the absence of Hoxb2, similarly to Hoxb1 mutant embryos. (G) E10.5 sagittal sections in dorsal regions indicate increased expression of Hoxa2 in ventricular (vz) and mantle (mz) zones of r4 (arrows, see also insets) mimicking the expression profile of r3. (H) Flat-mounted hindbrain preparations of E10.5 WT and Hoxa2^null embryos hybridized with Hoxb2. No particular expression changes can be observed in mutant embryos. Red arrowheads indicate a defect in alar r2/r3, as previously described . (I) Schematics summarizing the expression of Hox genes in r4 of Hoxb1 or Hoxb2 mutants. Scale bars, 200 µm (B, C, F and H); 100 µm (E top); 50 µm (E bottom). ba2, second branchial arc; me, mesoderm. See also , and .

(A) Strategy for the Cre-loxP recombinase r4-fate map. Upon Cre-mediated recombination, the loxP sites surrounding the PGKneo cassette of the ROSA26 YY reporter line are excised and YFP is expressed exclusively in r4 and r4 derivatives. (B) Dorsal and lateral views of a E10.5 b1r4-Cre/YFP embryo show restricted expression of YFP in r4 and neural crest-derived cells (ncc) in the second branchial arch (ba2). The white line delineates the level and plane of sagittal section of panels in (C). (C) r4-restricted immunostaining of Cre-recombinase in progenitors of the ventricular zone (vz). YFP⁺/Cre⁻ post-mitotic cells at the marginal zone (mz) originate from YFP⁺/Cre ⁺ progenitors. (D) Sagittal sections of an E12.5 b1r4-Cre/YFP embryo immunostained with a GFP antibody reveal the r4 domain, the caudal migration of facial branchiomotor neurons (mFBM), the ventricular to pial migration of presumptive lateral lemniscus cells (mVLL) and the lateral lemniscus tract (LLt) projecting rostrally. Below, a schematic of an E14.5 sagittal section indicating the position of the various nuclei. The red line delineates the plane of section of panels (E). (E) The olivocochlear (OC) neurons (delimitated by a red contour) express choline acetyltransferase (ChAT), Gata3 and Tbx20. (F) Schematic coronal section of a P8 brain illustrating the positions of the various nuclei. The lateral superior olive (LSO) but not the medial superior olive (MSO) nucleus, has an r4 origin, as indicated by YFP and VGlut2 staining. ChAT and Tbx20 are expressed in lateral (LOC) and medial OC (MOC) neurons within the LSO and ventral to the LSO, respectively. (G) Sagittal sections at different ages indicating the YFP⁺ r4-derivatives: the ventral lateral lemniscus (VLL) (rostrally) and FBM (caudally) nuclei. VLL and cochlear neurons project rostrally to the inferior colliculus (IC) and some fibers continue to the thalamus (arrowheads). (H) Coronal sections of P0 pups indicate high contribution of r4/YFP⁺ cells to the VLL, positive for Gad67, but not to the dorsal LL (DLL), which is VGlut2⁺. Hoxb2 and Hoxa2 are expressed in the VLL and pontine nuclei (PN). A few dispersed YFP⁺ cells are identified in the PN. (I) Schematic of a P8 brain sagittal section showing the position of the cochlear nuclear complex (CN) and its subdivision into anteroventral (AVCN), posteroventral (PVCN) and dorsal (DCN) nuclei. Adjacent sagittal sections illustrate a high contribution of YFP⁺ cells to the PVCN and DCN. The arrowhead indicates the origin of r4-migrating cells. Dots delineate the presumptive boundary between AVCN and PVCN. Only a few YFP-positive cells label the AVCN, which is highly positive for Atoh7. The small intensely basofilic granule cells confined to the microneuronal shell (mnsh and outlined) and identified by Nissl and Pax6 expression (J) are not positive for YFP, indicating that cochlear granule cells do not have an r4-origin. (K) Similarly, YFP⁺ cells do not co-localize with calbindin- (CB) and calretinin- (CR) expressing cells in the PVCN region. (L) Schematic of the hindbrain in which rhombomeres 2 to 5 and their respective Hox genes are color-coded. The same code refers to (M). (M) Overview of the two main central auditory pathways, the MOC reflex and their rhombomeric origin. While r4-derivatives (in red) contribute mainly to the ascending sound perception pathway, which runs from the CN through the VLL to the IC, r2-, r3- and r5-derivatives (in green) contribute mainly (but not exclusively) to the pathway running through the superior olivary complex (SOC), which function in the localization of the temporal and spatial origins of sounds. The MOC reflex comprising of sensory PVCN interneurons and motor efferent OC neurons is also an r4-derivative. Vn, trigeminal motor nucleus; MNTB, medial nucleus of trapezoid body; Pr5 principal sensory trigeminal nucleus. Scale bars, 100 µm (C), 200 µm (D–J left), 20 µm (J right, K). See also and .