The Distal Human myoD Enhancer Sequences Direct Unique Muscle-Specific Patterns of lacZ Expression during Mouse Development

Abstract

Transgenic mice carrying the bacterial lacZ reporter gene under the control of the regulatory elements of the human myoD gene have been produced. The developmental expression of the myoD reporter transgene in somites, limb buds, visceral arches, and cephalocervical regions was studied in transgenic embryos by beta-gal staining. In somites, the spatiotemporal pattern of transgene expression was different from other muscle-specific regulatory and structural genes and revealed that myoD-expressing cells arise in distinct patterns in somites that are dependent on position along the anterior-posterior (AP) body axis (occipital and cervical vs thoracic and more posterior myotomes). Transgene expression did not follow a strict anterior to posterior sequence of activation and therefore was not strictly correlated with somite developmental age. Moreover, the pattern of transgene expression along the dorsal-ventral myotomal axis was dependent on somite position along the anterior-posterior axis. While myoD expression is first detected after the myotome is well-formed, transgene expression in the dorsal and ventral medial lips of the dermatome suggests a function for myoD in the expansion of the myotome. Whole-mount in situ hybridization confirmed that these unique patterns of transgene expression in somites, as well as expression in limb buds, visceral arches, and other myogenic centers, are concordant with the distribution of endogenous myoD transcripts. These results shed new light on the developmental differences between myotomes at different positions along the AP and DV axis and demonstrate a unique axial pattern of somitic myoD expression, suggesting a specific role of myoD in myotome lineage determination and differentiation.

Two upstream enhancers collaborate to regulate the spatial patterning and timing of MyoD transcription during mouse development

“…b The specific regions of the CNS exhibiting expression varied between lines, although staining of the ventral neural tube was most common. All Ϫ24lacZ lines exhibited nasal pit expression, consistent with published observations for other core enhancer-containing transgenes (see Goldhamer et al, 1995;Faerman et al, 1995 Representative whole mount embryos from E9.75 to E12.5 comparing expression of lacZ in transgenic lines Ϫ24lacZ M22 (A-D) and Ϫ24⌬F3lacZ M57 (I-L) to in situ hybridization for endogenous MyoD (E-H). X-gal staining of a Ϫ24lacZ embryo at E9.75 (A) revealed lacZ expression in the mandibular arch (black arrow) and in the myotome of occipito-cervical somites (black arrowhead represents the first stained somite) to approximately the level of the forelimb bud (red arrowhead).…”

“…B: Section of E11.5 interlimb somite at high magnification. Transgene expression was occasionally observed in cells of the ventrolateral dermomyotomal lip (arrowhead), as previously described for a fragment 3-driven transgene (see Faerman et al, 1995). C: The Ϫ24⌬F3lacZ transgene was not expressed in the lateral, hypaxial myotome at E10.5 except, as shown here, for an occasional ␤-gal-positive cell that was observed in some embryos (arrowhead).…”

“…In contrast, MyoD mRNA was first detected by whole mount in situ hybridization in interlimb somites at E9.75 (initially in the hypaxial myotome) and subsequently in more anterior somites (present study; Tajbakhsh et al, 1997). Although we cannot formally rule out the possibility that the Ϫ24lacZ reporter construct lacks a regulatory element that normally represses early expression in anterior somites and branchial arches, this is unlikely since core enhancer-and DRRdriven transgenes also are not expressed at E9.5 (Asakura et al, 1995;Faerman et al, 1995;Goldhamer et al, 1995). Rather, expression of Ϫ24lacZ at E9.5 likely reflects cooperation between regulatory sequences in fragment 3 and the DRR (and perhaps other, unknown regulatory elements) combined with the greater sensitivity and resolution of ␤-gal histochemistry, and probably approximates the actual time at which MyoD transcription is initiated.…”

“…For esophagus, the core enhancer was used, for all others, fragment 3 was used. Data from Faerman et al (1995), except where noted. c All transgenes and MyoD mRNA (Faerman and Shani, 1993) were detected in the diaphragm rudiment at E11.5, except for mDRR-lacZ, for which data at E11.5 was not presented.…”

“…A second enhancer located 5 kb upstream of mouse MyoD, termed the distal regulatory region (DRR; Tapscott et al, 1992;Asakura et al, 1995), also drives expression of lacZ in a muscle-specific pattern in mouse embryos; however, DRR-lacZ transgenes show delayed expression in the limb buds and branchial arches. In addition, whereas a fragment 3 transgene and the endogenous MyoD gene are expressed prior to differentiation in limb and branchial arch lineages (Faerman and Shani, 1993;Faerman et al, 1995), DRR activity is restricted to differentiated cells (Asakura et al, 1995;Kablar et al, 1997). Furthermore, the DRR remains active in adult muscle, showing a similar expression profile to the endogenous MyoD gene (Asakura et al, 1995;Hughes et al, 1993), whereas fragment 3 and the core enhancer are down-regulated by late fetal stages and are essentially inactive in adult muscle Goldhamer et al, 1995).…”