Loss of the Sall3 gene leads to palate deficiency, abnormalities in cranial nerves, and perinatal lethality

Abstract

Members of the Spalt gene family encode putative transcription factors characterized by seven to nine C2H2 zinc finger motifs. Four genes have been identified in mice--Spalt1 to Spalt4 (Sall1 to Sall4). Spalt homologues are widely expressed in neural and mesodermal tissues during early embryogenesis. Sall3 is normally expressed in mice from embryonic day 7 (E7) in the neural ectoderm and primitive streak and subsequently in the brain, peripheral nerves, spinal cord, limb buds, palate, heart, and otic vesicles. We have generated a targeted disruption of Sall3 in mice. Homozygous mutant animals die on the first postnatal day and fail to feed. Examination of the oral structures of these animals revealed that abnormalities were present in the palate and epiglottis from E16.5. In E10.5 embryos, deficiencies in cranial nerves that normally innervate oral structures, particularly the glossopharyngeal nerve (IX), were observed. These studies indicate that Sall3 is required for the development of nerves that are derived from the hindbrain and for the formation of adjacent branchial arch derivatives.

FIG. 1.

Targeted disruption of Sall3. (A) (Top) Genomic structure of a portion of the Sall3 locus. (Middle) Targeting vector. (Bottom) Targeted locus. The majority of the coding sequence, including all zinc fingers, was removed and replaced with a LacZ-neomycin resistance cassette (LacZ/Neo). Grey bars indicate noncoding regions, brick-pattern bars indicate exons, stipple-pattern bars indicate LacZ/Neo, and black bars indicate locations of zinc finger motifs. E, B, S, H, and A, EcoRI, BamHI, SalI, HindIII, and ApaI restriction sites; parentheses indicate restriction sites that no longer exist. The locations of probes used for Southern hybridization (5′, LacZ, and 3′) are shown as black horizontal lines below the targeted locus. (B) Genotyping of Sall3 mutant animals. A 330-bp mutant band and the 220-bp wild-type band are shown. Samples from heterozygote animals yielded both bands. A positive control (+Ctl) and a negative control (−Ctl) are also shown. (C) RNase protection assay of a probe covering zinc fingers 4 and 5 indicating the absence of a Sall3 transcript in mutant animals. Lane m, labeled 1-kb marker; lane p, sizes of the unprotected Sall3 probe (394 bp) and the internal control TATA binding protein (TBP, which indicates that RNA was present in all samples) (210 bp); lane t, negative tRNA control. Other lanes show that mutant animals do not have a protected Sall3 band (330 bp), whereas wild-type and heterozygous animals do have a protected band.

FIG. 2.

Expression of Sall3 in developing craniofacial regions from E13.5 to E16.5. (A) Whole-mount β-galactosidase staining of a parasagittal section through a mutant E14 embryo. Staining is observed in the brain (b), heart (h), palate (arrow), and cranial nerves (arrowhead). (B and C) Magnified views of the hindbrain (B) and palate (C) regions from E14 embryos. Sall3 transcripts are detected in cochlear ganglia (c), in the glossopharyngeal ganglion (IX), in the posterior palate (pp), and in the anterior palate (ap). (D to F) Digoxigenin-labeled in situ hybridization in sagittal sections of E13.5 (D) and E16.5 (E and F) embryos demonstrating the patterns of expression of Sall3 (D and E) and Sall1 (F). Expression is evident in the posterior palate (red arrow), the frontonasal process (asterisk), and the developing tongue (t). Both Sall3 and Sall1 are expressed in the trigeminal (V), the vestibulocochlear (VIII), and the glosspharyngeal (IX) ganglia. In all sections, rostral is to the right and dorsal is to the left. The scale bar represents 2 mm for panel A, 750 μm for panels B and C, 350 μm for panel D, and 500 μm for panels E and F.

FIG. 3.

Oral structures are abnormal in Sall3^−/− animals. (A and B) Open-mouth views of whole-mount β-galactosidase staining of heterozygote (A) and mutant (B) embryos at P0. t, tongue; p, palate; asterisk, rugae. (C and D) Higher magnifications of the nasopharyngeal (np) and oropharyngeal (op) openings indicated by a red arrow in panel A. β-Galactosidase staining indicates that Sall3 is expressed throughout much of the palate, around the nasopharyngeal opening, and in the inner ear. In mutant animals (D), the nasopharyngeal opening is enlarged. (E to H) Cresyl violet staining of sagittal sections of tongues and palates of Sall3^+/+ (E and G) and Sall3^−/− (F and H) animals at P0 (E and F) and E16.6 (G and H). An abnormally large gap between the palate (velum) (black arrow) and the epiglottis (red arrow) is present in Sall3^−/− animals. The velum and the epiglottis are smaller in Sall3 mutant animals at both P0 (F) and E16.5 (H). The scale bar represents 3 mm for panels A and B, 1.3 mm for panels C to F, and 1 mm for panels G and H.

FIG. 4.

Shh expression and HNF3α expression in the first branchial arch and tongue are normal in Sall3^−/− animals. (A to H) Bright-field (A, B, E, and F) and dark-field (C, D, G, and H) photomicrographs of in situ hybridization of midsagittal sections from Sall3^+/+ and ^−/− animals for Shh at E9.5 (A to D) and HNF3α at E10.5 (E to H). The epithelium of the palate (arrows), the first branchial arch (b), and the esophagus are labeled with Shh (A to D) and HNF3α (E to H) in a similar manner in both wild-type and mutant animals. Rostral is to the left, caudal is to the right, dorsal is to the top, and ventral is to the bottom in each panel. fb, forebrain; hb, hindbrain. Scale bar, 200 μm.

FIG. 5.

Cranial nerve development is abnormal in Sall3^−/− animals at E10.5 to E11.5. (A to F) Whole-mount neurofilament staining of wild-type (A and C) and Sall3^−/− (B, D, E, and F) animals at E10.5 to E11.5. Rostral is to the top, caudal is to the bottom, dorsal is to the left, and ventral is to the right in each panel. Insets show higher magnifications of the areas of interest in each panel. (A) Wild-type embryo demonstrating the locations and normal morphology of cranial nerves (III, oculomotor; IV, trochlear; V, trigeminal; VII/VIII, facial/vestibulocochlear; IX, glossopharyngeal; X/XI, vagal/spinal accessory; XII, hypoglossal). (B and B inset) Sall3^−/− animal in which cervical roots that normally contribute to the hypoglossal (XII) nerve are truncated (arrow in inset). (C, D, C inset, and D inset) Bundles of fibers connecting the glossopharyngeal (IX) nerve with the trunks of the vagal and spinal accessory (X and XI) nerves in a wild-type animal (C) and, more conspicuously, in a Sall3^−/− animal (D) (arrows in insets). (E and E inset) Complete fusion of the glossopharyngeal (IX) and vagal/spinal accessory (X/XI) trunks in a Sall3^−/− animal (arrow in inset). (F and F inset) Example in which the glossopharyngeal inferior (IX) ganglion lacked a connection to the brain stem (arrow in inset). The scale bar represents 800 μm for panel A and 500 μm for panels B to F.

FIG. 6.

The hypoglossal nucleus is normal in Sall3^−/− animals. (A to D) Neurofilament staining of coronal sections demonstrates the normal location and appearance of the hypoglossal nucleus (XIIn) (perimeter highlighted) in Sall3^+/+ (A and C) and Sall3^−/− (B and D) animals. 4V, fourth ventricle; small arrows, axons. Dorsal is to the top and ventral is to the bottom in each panel. The scale bar represents 500 μm for panels A and B and 200 μm for panels C and D.

FIG. 7.

Early expression of transcription factors in the spinal cord is normal in Sall3^−/− animals. (B, C, E, and F) Coronal sections of the spinal cord at E12.5 show the expression of HNF3α RNA in the floor plate (B and C; black grains; red arrowheads) and Pax6 RNA (E and F; white arrows; dark-field images) in Sall3^+/+ (B and E) and Sall3^−/− (C and F) animals. The gross patterning of the spinal cord in Sall3^−/− animals (C and F) is similar to that in wild-type animals (B and E). (A and D) Bright-field images of Sall3^+/+ spinal cords are included for reference adjacent to the experimental sections (B, C, E, and F). Dorsal is to the top and ventral is to the bottom in each panel. The scale bar represents 300 μm for all panels.